Post on 29-Dec-2018
BASIS OF HOST PLANT RECOGNITION AND
ACCEPTANCE BY BUSSEOLA FUSCA (FULLER)
(LEPIDOPTERA: NOCTUIDAE) LARVAE
GERALD JUMA
DOCTOR OF PHILOSOPHY
(Biochemistry)
JOMO KENYATTA UNIVERSITY OF
AGRICULTURE AND TECHNOLOGY
2010
Basis of host plant recognition and acceptance by Busseola
fusca (Fuller) (Lepidoptera: Noctuidae) larvae
Gerald Juma
A Thesis Submitted in Fulfilment for the Degree of Doctor
of Philosophy in Biochemistry in the Jomo Kenyatta
University of Agriculture and Technology
2010
ii
DECLARATION
This thesis is my original work and has not been presented for a degree in any other
university.
Signed…………..……..................... Date....................................
Gerald Juma
This thesis has been submitted for examination with our approval as university
supervisors.
Signed…………..……..................... Date....................................
Prof. Gabriel Magoma
JKUAT, Kenya
Signed…………..……..................... Date....................................
Dr. Paul-André Calatayud
IRD / ICIPE- Kenya
Signed…………..……..................... Date....................................
Dr. Bruno Le Rü
IRD / ICIPE-Kenya
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DEDICATION
This piece of work is dedicated to my lovely mum, Kalasina, my dear wife
Carolyn, my lovely children, Faith and Ken and my late brother Kenneth.
To mum and Kenneth: Thank you for your love, care, endurance and prayers
To Carolyn, Faith and Ken: You’re the source of my inspiration.
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ACKNOWLEDGMENTS
I would like to sincerely and warmly thank all the persons who participated in the
achievement of this work. Specifically, I acknowledge with gratitude my sponsors,
Institut de Recherche pour le Développement (IRD) and International Centre of
Insect Physiology and Ecology (ICIPE) for the financial support they provided for
this research. I sincerely thank Jomo Kenyatta University of Agriculture and
Technology (JKUAT) for having granted me the opportunity to pursue my studies
and for providing me with the ever encouranging and ready to assist academic
mentors. I wish to specially thank Dr. Paul-André Calatayud for granting me the
chance to conduct my PhD research project, for his immense contribution to this
work, constructive advice and critically reviewing the manuscript as well as for his
encouragements and support during difficult moments.
I wish to sincerely thank my supervisors Prof Gabriel. Magoma and Dr Bruno LeRü
for the deep interest they took in this work and for the immense guidance and
critical discussion rendered during various stages of my study period. I also extend
my sincere and warm thanks to Dr. Baldwin Torto for allowing me to conduct my
work in his laboratory, members and all my fellow students in the Chemical
Ecology department for all the rich discussions that we had during my research
work. Special thanks go to Dr. Njagi for his guidance in carrying out
electrophysiological tip recording experiments, Mathayo Chimtawi for his guidance
in scanning electron microscopy and Anthony Wanjoya in my data analysis. A lot of
appreciation goes to Mr. Peter Ahuya, Edward Nyandat, John Angira, Onesmus
Wanyama, Peter Malusi and Edwin Rono for their technical support.
v
I am indeed grateful to Mr. George Ong’amo for his assistance in the molecular
characterisation and Margret Thiong'o and Laure Dutaur of ICRAF for their
immense and tireless efforts in assisting in HPLC analyses of my samples and Mr.
Wambua of Geology Department for his technical support rendered in the
determination of silica in my plant samples. Special thanks go to all colleagues and
staff of Chemical Ecology, Biodiversity, Bioprospecting, Molecular Biology and
Biochemistry units for their assistance, support, cooperation and encouragement
rendered during my research work. Special thanks go to the members of my jury
who kindly accepted to judge my work.
My deepest gratitude go to my family, first my wife Carolyn, our daughter, Faith
and son Ken, who endured periods of my prolonged absence from home and for
their patience, love and unfailing encouragement. I remain indebted to my mother,
my siblings and friends who have been there for me all this trying time. To all who
are not mentioned but in one way or another contributed towards the successful
completion of this work, I say a big thank you.
Finally, I am grateful to the almighty God for giving me the courage, wisdom,
strength and motivation and enabling me to complete this work successfully and
within stipulated time.
Thanks to all and God bless you.
vi
TABLE OF CONTENTS
DECLARATION ......................................................................................................II
DEDICATION .........................................................................................................III
ACKNOWLEDGMENTS.......................................................................................IV
TABLE OF CONTENTS ........................................................................................VI
LIST OF TABLES.................................................................................................... X
LIST OF FIGURES ...............................................................................................XIII
LIST OF APPENDICES ........................................................................................XV
LIST OF ABBREVIATIONS ..............................................................................XVII
ABSTRACT ...........................................................................................................XIX
CHAPTER ONE ........................................................................................................1
1. 0: INTRODUCTION AND LITERATURE REVIEW .......................................1
1. 1: GENERAL INTRODUCTION ...................................................................................1
1. 2: RATIONALE ...........................................................................................................5
1. 3: GENERAL OBJECTIVE............................................................................................7
1.3.1: SPECIFIC OBJECTIVES..........................................................................................7
1. 4: L ITERATURE REVIEW ..........................................................................................8
1. 4. 1: HOST RANGE AND SHIFT IN PHYTOPHAGOUS LEPIDOPTRAN INSECTS.................8
1. 4. 2: INFLUENCE OF PLANT CHEMISTRY IN HOST CHOICE.........................................11
1. 4. 3: PHYLOGENETIC CONSTRAINTS TO HOST USE....................................................14
1. 4. 4: PHYSIOLOGICAL CONSTRAINTS TO HOST USE..................................................17
1. 4. 5: “L EARNING” AS A MECHANISM OF HOST SWITCH.............................................18
1. 4. 6: ECOLOGICAL CONSTRAINTS TO HOST CHOICE..................................................20
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1. 4. 7: INFLUENCE OF PLANT PHYSICAL ATTRIBUTES TO HOST CHOICE.......................20
1. 4. 8: NEURAL CONSTRAINTS TO FOOD CHOICE.........................................................22
CHAPTER TWO.....................................................................................................25
2.0: DISTRIBUTION OF CHEMO AND MECHANORECEPTORS ON THE
ANTENNAE AND MAXILLAE OF B. FUSCA LARVAE...........................25
2. 1: INTRODUCTION ..................................................................................................25
2. 2: MATERIALS AND METHODS..............................................................................26
2. 2. 1: INSECT REARING.............................................................................................26
2. 2. 2: SCANNING ELECTRON MICROSCOPY................................................................28
2. 2. 3: SILVER NITRATE STAINING ..............................................................................28
2. 2. 4: ELECTROPHYSIOLOGY.....................................................................................29
2. 2. 5: DATA ANALYSIS ..............................................................................................30
2. 3: RESULTS ............................................................................................................30
2. 3. 1: SENSILLA PRESENT ON THE LARVAL ANTENNAE..............................................30
2. 3. 2: SENSILLA PRESENT ON THE LARVAL MAXILLAE ..............................................34
2. 4: DISCUSSION .......................................................................................................38
CHAPTER THREE.................................................................................................42
3.0: EFFECT OF HOST PLANT’S METABOLITES AND SILICA ON
SURVIVAL AND GROWTH OF B. FUSCA LARVAE................................42
3. 1: INTRODUCTION ..................................................................................................42
3. 2: MATERIALS AND METHODS..............................................................................44
3. 2. 1: INSECTS..........................................................................................................44
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3. 2. 2: PLANTS ...........................................................................................................45
3. 2. 3: SURVIVAL AND GROWTH OF B. FUSCA LARVAE ON DIFFERENT PLANT SPECIES45
3. 2. 4: PREPARATION AND EXTRACTION OF PLANT LEAF POWDERS............................46
3. 2. 5: FEEDING PREFERENCE TESTS...........................................................................47
3.2.6: ANALYSIS OF SIMPLE SUGARS IN PLANT LEAF EXTRACTS..................................51
3.2.7: ANALYSIS OF TOTAL PHENOLIC COMPOUNDS IN PLANT LEAF EXTRACTS...........52
3.2.9: EXTRACTION AND ANALYSIS OF SILICA IN LEAVES OF DIFFERENT PLANT SPECIES...53
3.2.10: SURVIVAL AND GROWTH OF LARVAE ON SILICA AMENDED DIETS...................55
3.2.11: STATISTICAL ANALYSIS ..................................................................................56
3.4: RESULTS.............................................................................................................57
3.4.1: EFFECT OF HOST PLANTS ON THE SURVIVAL AND GROWTH OF B. FUSCA LARVAE .....57
3.4.2: INFLUENCE OF PLANT METABOLITES ON THE PERFORMANCE OF B. FUSCA
LARVAE ...........................................................................................................60
3. 4.3: INFLUENCE OF PLANT SILICA ON SURVIVAL AND GROWTH OF B. FUSCA LARVAE ....71
3. 5: DISCUSSION .......................................................................................................75
CHAPTER FOUR ...................................................................................................83
4. 0: PHYSIOLOGICAL ADAPTATIONS AND GENETIC DIVERSITY OF B.
FUSCA LARVAE IN RELATION HOST PLANT USE...............................83
4. 1: INTRODUCTION ..................................................................................................83
4. 2: MATERIALS AND METHODS..............................................................................86
4. 2. 1: INSECTS...........................................................................................................86
4. 2. 3: DETERMINATION OF ACTIVITIES OF DIGESTIVE ENZYMES IN LARVAL
HOMOGENATES OF LARVAE FED ON LEAVES OF DIFFERENT PLANT SPECIES.....87
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4. 2. 4: DNA ISOLATION AND PURIFICATION...............................................................89
4. 2. 5: CYTOCHROME B GENE AMPLIFICATION AND DIGESTION..................................90
4. 2. 6: CYTOCHROME B GENE SEQUENCING AND ANALYSIS........................................91
4. 3: RESULTS ............................................................................................................92
4. 3. 1: ACTIVITIES OF DIGESTIVE ENZYMES IN LARVAL HOMOGENATES.....................92
4. 3. 2: POPULATION DISTRIBUTION ............................................................................96
4. 4: DISCUSSION .......................................................................................................99
CHAPTER FIVE ...................................................................................................107
5. 0: GENERAL DISCUSSION, CONCLUSION AND RECOMENDATIONS ...107
5. 1: GENERAL DISCUSSION.....................................................................................107
5. 2: CONCLUSION AND RECOMMENDATIONS .........................................................113
6. 0: REFERENCES ..............................................................................................119
7. 0: APPENDICES................................................................................................159
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LIST OF TABLES
Table 2.1: Amount of ingredients used to prepare a litre of artificial diet used to
rear Busseola fusca larvae (Onyango and Ochieng’-Odero, 1994)...27
Table 2.2: Meana lengths (µm, ± SE; n=4) of antennal sensilla of Busseola fusca
neonate and third instar larvae...........................................................31
Table 2.3: Meana lengths (µm, ± SE; n=4) of maxillae sensilla of neonate and
third instar larvae of Busseola fusca..................................................35
Table 3.1: Percentage of surviving (mean1 ± SE, n=12) and growth rates (mg d-
1, mean2 ± SE) of Busseola fusca larvae recovered after 7, 19 and 31
days of infestation by neonates on different Poaceae plant species..59
Table 3.2: Percentages of surviving third instar Busseola fusca larvae recovered
(mean ± SE, n=8, except for Z. mays and S. arundinaceum n=7) and
growth rates (mg d-1, mean ± SE) after 7 and 19 days of infestation
on different plant species...................................................................60
Table 3.3: Feeding response indices (%, mean ± SE, n=30) for third instar
Busseola fusca larvae after 36 hours of feeding on crude leaf extracts
of different plant species topically applied on Styrofoam carrier
matrices under no-choice conditions.................................................62
Table 3.4: Feeding response indices (%, mean ± SE, n=30) for third instar
Busseola fusca larvae after 36 hours of feeding on crude leaf extracts
of different plant species topically applied on Styrofoam carrier
matrices under dual choice conditions..............................................63
Table 3.5: Feeding response indices (%, mean ± SE, n=30) of third instar
Busseola fusca larvae after 36 hours of feeding on crude leaf extracts
xi
of different plant species topically applied on Styrofoam carrier
matrices under 4-choice conditions...................................................64
Table 3.6: Feeding response indices (%, mean ± SE, n=87) of third instar
Busseola fusca larvae after 36 hours of feeding on crude methanol
extracts of different plant species topically applied on styrofoam
carrier matrices under 8-choice conditions........................................65
Table 3.7: Amount of different sugars (µg/mg of leaf dry weight, mean ± SE,
n=5) found in leaves of the plant species studied..............................67
Table 3.8: Relative proportion of sugars (%, mean ± SE, n=5) found in the dry
leaves of the plant species studied.....................................................68
Table 3.9: Content of total phenolic compounds (in µg gallic acid
equivalents/mg of dry leaf weight, mean ± SE, n=10) found in leaves
of the plant species studied................................................................69
Table 3.10: Feeding response indices (%, mean ± SE, n=24) for third instar
Busseola fusca larvae after 36 hours of feeding on mixture of sucrose
and turanose of varying concentration topically applied on styrofoam
carrier matrices under no-choice (A) and 6-choice conditions (B)...71
Table 3.11: Amount of silica (mean ± SE, n=34) found in different Poacea plant
species used in the study....................................................................72
Table 4.1: Semi-quantitative analysis of enzyme activities in the homogenate of
entire neonate larvae (with decapitated heads) reared for 4 days on
different plants species. Enzyme activities (means ± SE, n=5)
correspond to the activities found in the crushed larval homogenate
(40 larvae per replicate). Activities correspond to the release of 5, 10
xii
and 20 nmoles of substrate per 4 hours of incubation at 37°C (rate 0:
no change in activity)........................................................................94
Table 4.2: Correlations between enzyme activities and percentages of surviving
larva of Busseola fusca recovered or relative growth rates (RGR)
after 7, 19 or 31 days, following infestation by neonates. R-values
are given............................................................................................95
Table 4.3: Summary of the distribution of Busseola fusca clades with respect to
host plant species and locality...........................................................98
Table 4.4: Genetic diversity (mean ± SD) of the cytochrome b gene of Busseola
fusca sampled from Z. mays and A. donax from Kenya and Ethiopia
respectively............................................Error! Bookmark not defined.
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LIST OF FIGURES
Figure 2.1: Antenna of third instar Busseola fusca larva.....................................32
Figure 2.3: Dose-response curves for sucrose obtained after contact with the
cone-shaped basiconic sensillum on the third antennal segment (A)
and with the maxillary palp (B) of third instar larva. ........................33
Figure 2.4: Maxilla of third instar Busseola fusca larva ......................................36
Figure 2.5: Contact electrophysiological recording of spike activity after contact
with a lateral sensillum styloconicum on the maxillary galea of a
third instar larva.................................................................................37
Figure 3.1: An illustration showing the preparation of Styrofoam cylinders (13
mm diameter, 35 mm long) used as feeding matrix for third instar
Busseola fusca larvae to which plant extracts were applied during
larval feeding bioassay. .....................................................................48
Figure 3.2: Illustrations for bioassay feeding setups for third instar Busseola
fusca larva: single choice (A) dual choice (B) 4-choice (C) and 8-
choice feeding tests (D)……. ............................................................50
Figure 3.3: Dose-response curves for relative feeding indices (mean ± SE, n=15)
obtained by increasing sucrose (A) or turanose (B) levels under no-
choice conditions. ..............................................................................70
Figure 3.4: Dose-response curves for percentage of surviving larvae recovered
(arcsin transformed values, mean ± SE, n=9) (A) and relative growth
rates (RGR) (mg.d-1, mean ± SE, n=9) (B) obtained after increasing
silica concentration. ...........................................................................74
xiv
Figure 4.1: A representative plate showing differential staining of the API ZYM
plate with ZYM A and ZYM B reagents after 4 hours of incubation
of homogenates of larvae fed on plant leaves with chromogenic
substrates ...........................................................................................93
Figure 4.2: A representative agarose gel electrophoresis plate of DNA products
of cytochrome b DNA fragment of several Busseola fusca larvae
randomly sampled from Z. mays and A. donax noted from 1 to 9
following PCR amplification and Xho restriction digestion. Distinct
bands were identified using 100bp ladder and results showed the
presence of two mitochondrial clades labelled KI and KII. ...............96
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LIST OF APPENDICES
Appendix A: Representative HPLC Chromagram obtained following injection
of 2 µL solution of 10mg/ml dried Zea mays methanol extracts
reconstituted in water into a supelcosil LC-NH2 column in
Shimdzu class-VP auto sampler machine….…………………159
Appendix B: Representative HPLC Chromagram obtained following injection
of 2 µL solution of 50mg/ml dried Sorghum arundinaceum
methanol extracts reconstituted in water into a supelcosil LC-
NH2 column in Sheds class-VP auto sampler machine……....160
Appendix C: Representative HPLC Chromagram obtained following injection
of 2 µL solution of 50mg/ml dried Setaria megaphylla methanol
extracts reconstituted in water into a supelcosil LC-NH2 column
in Shimdzu class-VP auto sampler machine………………….161
Appendix D: Representative HPLC Chromagram obtained following injection
of 2 µL solution of 50mg/ml dried Panicum desteum methanol
extracts reconstituted in water into a supelcosil LC-NH2 column
in Shimdzu class-VP auto sampler machine………………….162
Appendix E: Representative HPLC Chromagram obtained following injection
of 2 µL solution of 50mg/ml dried Panicum maximum methanol
extracts reconstituted in water into a supelcosil LC-NH2 column
in Shimdzu class-VP auto sampler machine………………….163
xvi
Appendix F: Representative HPLC Chromagram obtained following injection
of 2 µL solution of 50mg/ml dried Arundo donax methanol
extracts reconstituted in water into a supelcosil LC-NH2 column
in Shimdzu class-VP auto sampler machine………………….164
Appendix G: Representative HPLC Chromagram obtained following injection
of 2 µL solution of 50mg/ml dried Peniseteum purpureum
methanol extracts reconstituted in water into a supelcosil LC-
NH2 column in Shimdzu class-VP auto sampler machine…...165
Appendix H: Representative HPLC Chromagram obtained following injection
of 2 µL solution of 12mg/ml mixture of sugar standard solutions
reconstituted in water into a supelcosil LC-NH2 column in
Shimdzu class-VP auto sampler machine………………….....166
xvii
LIST OF ABBREVIATIONS
AC/DC Alternate current/ Direct current
ANOVA Analysis of Variance
ARCU Animal Rearing and Containment Unit (ARCU)
CNRS National Centre of Scientific Research
Cyt b Cytochrome b
DIMBOA 4-dihydroxy-7-methoxy-1, 4-benzoxazin-3-one
DNA Deoxyribonucleic Acid
ECB European corn borer
EDTA Ethylenediaminetetraacetic acid
FRI Feeding Response Index
GLM General Linear Model
HF Hydrofluoric acid
HCl Hydrochloric acid
HPLC High Pressure Liquid Chromatography
ICIPE International Centre for Insect Physiology and Ecology
ICRAF International Centre for Research in Agro-Forestry
IRD Institut de Recherche pour le Développement
KI Kenyan 1
KII Kenyan II
L/D Light/ Dark photoperiod proportion
LS Lateral Styloconicum
MFO Microsomal Function Oxidases
MS Medial Styloconicum
xviii
NCPA Nested Clade Phylogeographical Analysis
PCR Polymerase Chain Reaction
PTFE Polytetrafluoroethylene (Teflon)
PLSD Protected Least Significant Difference.
PVP Polyvinyl-pyrollidone
RGR Relative Growth Rate
Rh Relative humidity
SE Standard Error
SD Standard Deviation
SAS Statistical Analysis Software
SDS Sodium dodecyl sulphate
Taq Thermus aquaticus
UV Ultra-Violet
xix
ABSTRACT
Busseola fusca (Fuller) (Lepidoptera: Noctuidae) is an important pest of maize and
sorghum in sub-Saharan Africa whose larvae exhibits oligophagic than polyphagic
feeding habits. The host plants for this species are primarily maize and sorghum
though some populations appear to be restricted on wild sorghum. The purpose of
this study was to determine the extent of oligophagy of larvae of this species based
on (i) the sensory abilities to discriminate among different host plants (ii) feeding
behaviour and growth on different Poaceae plant species present in its natural
habitat (iii) plant stimuli that influence larval growth and feeding and (iv) the
physiological adaptations to various host plant diets. The potential existence of
genetically determined host-plant associated populations was also investigated. The
results obtained from scanning microscopic preparations, selective silver nitrate
staining and dose response electrophysiological experiments indicate that larval
sensory structures present on the maxillae and the antennae are typical of other
lepidopteran species and consist mainly of multiporous olfactory and uniporous
gustatory sensilla. These sensorial equipments are thought to be involved in
discriminating amongst chemical cues important for larval host recognition and
selection. The gustatory role of both sensorial equipments was confirmed from the
significant and positive dose-response electrophysiological tip recording tests, for
the antennal sensillum (F1,56 = 41.637, P<0.0001) and for the maxillary palp (F1,58 =
32.124, P<0.0001) using increasing concentrations of sucrose. Moreover, this study
demonstrated for the first time the presence of antennal taste receptors on a
lepidopteran larva key to host choice, explaining the ability of larvae to quickly
evaluate the phago-suitability of the host plant following landing on its surface.
xx
Among the selected Poacaeae plant species used and which form the natural feeding
repertoire of B. fusca, only Zea mays and Sorghum arundinaceum supported the
highest larval performance. Endogenous silica which is thought to negatively
influence feeding behaviour of many herbivorous insects, varied significantly
among the plant species studied. The amount of silica found among the plant
extracts studied ranged between 20µg/mg-55µg/mg of dry leaf weight following
spectrophotometric determination of each of the plant leaf samples digested with
dilute hydrofluoric acid. The silica levels in the plants’s digests correlated
negatively with both larval feeding and growth rates, hence confirming the
importance of silica as a significant barrier to dietary adaptation by B. fusca larvae.
All the polar and methanol-soluble plant extracts tested elicited feeding of third
instar larva as compared to the non-polar hexane extracts both in choice and in non-
choice bioassays. Methanol extracts of Z. mays, S. arundinaceum and Arundo donax
were however the most phagostimulatory. Plant sugar content as identified by HPLC
also varied among plants tested with sucrose contributing the highest (22µg/mg to
65µg/mg of dry leaf weight), which was an equivalent of between 25%-38% of the
total sugar content of each plant leaf extract analysed. Larval feeding positively
correlated with sucrose content but negatively with turanose, the most variable sugar
fraction (5-36µg/mg dry leaf weight) of the plant extracts analysed. However,
additional bioassays indicated that whereas turanose and sucrose play a
phagodeterrent and phagostimulant roles respectively, a balance between the two
(probably in the ratio of 3:1 respectively) appeared to be an important factor in host
acceptance for larval feeding. Therefore, the level of silica and the balance between
xxi
the two sugars in the plant leaves seem to be key determinants of host plant choice
and acceptance for feeding and growth by B. fusca larvae.
Larvae fed on Z. mays and S. arundinaceum; plants that supported the highest larval
survival and growth rates had pronounced levels of sugar than amino acid degrading
enzymes, indicating the general over reliance of larvae on carbohydrates over
proteins for survival. However, esterase enzymes were also highly induced in
homogenates of larvae that consumed leaves of their least preferred host plants that
included P. maximum P. desteum and S. megaphylla. These three plant species had
also the highest total phenolic content in their leaf tissues following
spectrophotometric determination of the same in their leave tissues. Therefore, the
induction of esterase in larvae fed on the three high phenolic containing plants could
possibly indicate the accompanying physiological responses of larvae to a specific
or a number of deterrent phenolic glycosides in the particular plant leaf tissue.
Finally, no conclusive evidence was deduced for host associated genetic
differentiation among individuals of B. fusca larva found on wild plants (A. donax)
or cultivated crops (Z. mays) as was inferred from genetic analysis of two fragments
of the gene coding for cytochrome b.
1
CHAPTER ONE
1. 0: INTRODUCTION AND LITERATURE REVIEW
1. 1: General introduction
In sub-Saharan Africa, phytophagous lepidopteran stem borers are among the major
field insect pests that cause extensive damage to cereal crops, particularly maize and
sorghum (Ingram, 1958; Seshu Reddy, 1991). Yield losses caused by stemborer
pests are reported to vary widely among regions depending on the pest population
density and phenological stage of the crop at infestation. Estimates of crop losses
due to stemborer damage in sub-Saharan Africa varies from 20 to 40% of the
potential yield (Seshu Reddy and Walker, 1990; DeGroote, 2002), indicating the
importance of phytophagous stem borers as a major limiting factor of cereal crops
productivity in the region. Most cereal stem-borers of maize and sorghum are
generally polyphagous and have several other cultivated and non-cultivated
graminaceous host plants (Khan et al., 1991).
Busseola fusca (Fuller) (Lepidoptera: Noctuidae), a phytophagous noctuid moth
indigenous and restricted to Africa, is among the most economically important and
widely spread stemborer pest of cereals in the sub-Saharan Africa (Harris and
Nwanze, 1992; Kfir et al., 2002). Third instars larvae of B. fusca always bore and
feed inside the stems of host plants (grasses and cereal crops) especially maize and
sorghum and therefore cause major yield losses in subsistence cereal production
throughout sub-Saharan Africa. The percentage loss of cultivated cereals accruing
from damage by B. fusca alone is estimated to range between 10 and 39% in
2
southern Africa (van den Berg and Ebenebe, 2001) and 4-73% in eastern Africa
(Seshu Reddy and Walker, 1990).
Larva which is the most destructive stage of this pest initially feeds on host plant
leaves but later, during its third instar stage, penetrates the stems and cause
extensive damage to the infested plants as a consequence of feeding and tunnelling
into the plant stem.
B. fusca is primarily a pest in the dry savannah zone of Africa with its distribution
status varying by region. It occurs throughout mainland Africa, south of the Sahara
and has been formally recorded in West, East and Southern Africa (Harris &
Nwanze, 1992). In West Africa, the species occurs from sea level to an altitude of in
excess of 2000 M including the wetter parts of the tree savannah of Ghana (Tams &
Bowden, 1953) and Burkina Faso (Nwanze, 1988). In Eastern and southern Africa,
B. fusca is generally a pest of higher altitudes, occurring at altitudes of between 600
and 2700 M (Sithole, 1989). In Southern Africa, B. fusca is the dominant stemborer
species at altitudes above 900 M but some populations have been found at a much
lower altitudes in the same region, indicating the ability of this species to adapt to
lowlands and warmer areas (Sithole, 1989). Similarly, some B. fusca populations
have been recently recovered from the coastal areas of Kenya and Tanzania
contradicting reports that previously indicated the absence of this species in coastal
regions (Harris and Nwanze, 1992), but similarly suggesting the ability of the B.
fusca to spread out in more areas than initially reported.
A Phylogenetic and Nested Clade Phylogeographical (NCPA) genetic analysis has
separated B. fusca into three geographically isolated population units: one from
Western Africa, and two from Eastern and Central Africa (Sezonlin et al., 2006).
3
These three population units display ecological preferences along altitudinal
gradients with the West African population primarily inhabiting the low altitudes of
dry savannah zones (Harris and Nwanze, 1992). The East and central Africa
populations predominantly inhabit the wet and cold zones of the highlands (Kfir et
al., 2002). The intra and inter-population distance observed among the populations
however suggest that these three population units were isolated at the same period
into three different refuges of sub-Saharan Africa prior to human-mediated
ecological changes as a result of the climatic and /or geological events and thus
suggest geographical isolation of the species (Sezonlin et al., 2006).
Like many other local phytophagous species, B. fusca is hypothesized to have
coevolved with indigenous wild graminaceous plants of Sub-saharan Africa, before
the domestication of sorghum and the introduction of maize into Africa (Ristanovic,
2001). However, from field data, it appears that the species has had a drastic
divergence in host colonization; shifting from ancestral grasses to important
cultivated staple cereal crops of the region over the last centuries (Sezonlin et al.,
2006). For example, recent field studies on the species host range indicate that B.
fusca is localized and specialized on a narrow range of host plant species especially
cultivated maize and sorghum (Le Rü et al., 2006a, 2006b; Ong’amo et al., 2006).
Moreover, marginal utilisation of wild poaceous plants has also been documented
for the species in some regions. In the wild, a significant number of young larvae
have been recovered from wild sorghum, Sorghum arundinaceum (Desv.) Stapf, and
Arundo donax L. in a number of localities in Eritrea, Ethiopia and South Africa. (Le
Rü et al., 2006). Anecdotal recoveries have also been made on Pennisetum
purpureum (Schumach.) and Cymbopogon nardus (L.) Rendle, in eastern Africa,
4
particularly in Kenya and Tanzania as well as on Setaria megaphylla (Steud.) T.
Duran & Schinz, in Congo in central Africa (Le Rü et al. 2006b). These surveys
clearly points to the oligophagic feeding habits of B. fusca larvae. Moreover, patchy
utilisation of different wild grasses may also point to the possible existence of host
plant specific strains within B. fusca populations (Le Rü et al., 2006a, Sezonlin et al.
2006), although little information is available regarding the performance traits of
the wild populations on the associated wild grasses to corroborate this hypothesis.
The mechanisms of host plant selection and recognition for oviposition sites have
been studied for the adults of B. fusca (Juma, 2005), but similar information on
larval feeding behaviour has largely been unexplored. For B. fusca larvae, the
recognition and subsequent choice of host plant for feeding can be hypothesised to
partially depend on host plant chemical composition and / or physical
characteristics. Nevertheless, morphological differentiation among insect’s
populations may also be a consequence of different diet breadths. Host correlated
morphological differentiation in relation to host choice and use has been described
among several insects’ species (Langor and Spence, 1991; Bernays, 1991; Bernays
2001). For example, in sap-sucking insects, variations in the lengths of mouthparts,
leg segments and the number of sensilla have been associated with different host
plant use (Carroll and Boyd, 1992; Bernays et al., 2000; Margaritopoulos et al.,
2000). These findings support the theory of adaptability of insect morphology to
differential plant use, suggesting that wider diet breadth should be correlated with
higher morphological variation in structures involved in host selection and feeding.
However, given the importance of host plant selection process prior to larval
adoption and the variable behavioural feeding responses during development, it is
5
likely that B. fusca larvae have special structures of high morphological variation
among its developmental stages that ensure efficient detection of wide range of plant
stimuli that mediate host recognition and subsequent selection of feeding sites.
Elucidating how recognition, selection and constraints that affect the emergence of
host choice is therefore important to bring into focus the mechanism by which B.
fusca switched from its ancestral wild grass to cultivated hosts to become an
economically important pest of several cultivated cereal crops. The purpose of this
study was therefore to understand the basis of host plant selection and acceptance
for feeding by B. fusca larvae. This data is important for the development of future
control and management strategies of this most destructive pest of cereal crops in
the Sub-saharan Africa.
1. 2: Rationale
Wild grasses (for example wild sorghum, Sorghum arundinaceum [Desv.] Stapf
[Poaceae]) were probably the native host plants of B. fusca prior to the
domestication of sorghum and the introduction of maize into Africa. B. fusca which
is currently the most economically important pest of maize is speculated to have
shifted from the native wild grasses to closely related exotic plants, including Zea
mays L. [Poaceae]) ultimately expanding its host range. However, the mechanism(s)
underlying this host switch is still unknown. Ecological availability of new crops in
the field, insect physiological adjustments (adaptive plasticity), efficient sensory
system and/or genetic selection pressures are speculated to have heavily contributed
to the switch. The existence of different geographic and possibly host plant specific
strains have been postulated by Sezonlin et al. (2006). Such candidate host plant
6
associated strains have been reported to inhabit Arundo donax L. (Poaceae) found in
Ethiopia, Eritrea and more recently, South Africa (Le Rü, 2006b). These conspecific
strains seem to show conservatism in feeding on wild grasses suggesting host plant
specificity.
Understanding the evolution of host or habitat specialisation in B. fusca has
important implications not only for evolutionary ecology, but also for the
development of sound control strategies. For example, a novel stimulo-deterrent
diversionary or the ‘push-pull’ strategy (Miller and Cowles, 1990) has been
developed in East Africa as an integrated pest management approach against B.
fusca and other stemborer pests (Khan et al., 2000; Khan and Picket, 2004; Cook et
al., 2007). The approach exploits the underlying chemical defense mechanisms of
trap crops planted as border rows (pull), and repellent and unsuitable plant species
(push) intercropped with the cereal crops. At present it is impossible to determine
whether in future B. fusca might evolve rapidly in host range and adapt to non target
plants currently used as repellent intercrops in the push-pull strategy and therefore
adversely affect the sustainability of this promising management strategy. Although
some of the plants used in this approach are not closely related to the species normal
plant diets, it is speculative that as a result of repeated exposure of the pest to these
plants, this pest may undergo evolutionary or elicit adaptive changes that may
involve expansion of its diet breadth and include non target plants important in this
strategy, thereby negatively affecting its management and control.
The current study therefore, sought to infer the recognition, selection, feeding,
development and performance patterns of B. fusca larvae on various hosts and
candidate host plants. Since the likelihood of rapid evolution of shift to a non-target
7
plant may be judged to some extent by screening populations of the insect for
genetic variations and behavioural responses, the genetic structure of the candidate
host associated B. fusca populations was also studied to infer the correlation
between genetic structure and host use, and to provide a framework for the
understanding of the mechanisms involved in novel host-plant adoption by larvae of
this species. The following questions were thus addressed.
1. Is B. fusca larvae able to recognise suitable host plant for colonisation?
2. Which plant factors influence larval development and performance?
3. Do larvae physiologically adapt to chemotaxonomically variable plant diets
encountered during its oligophagic foraging habits.
4. Does host use influence genetic sub-structuring of B. fusca populations?
1. 3: General objective
This study was undertaken with a broad objective to asses the effects of host plant’s
chemical and physical attributes on feeding behaviour and performance and to
determine the physiological and genetic basis of host plant preference by B. fusca
larva.
1.3.1: Specific objectives
The specific objectives of this study were:
1. To determine larval sensory structures important in host plant recognition and
acceptance;
2. To verify the extent of feeding and growth of B. fusca larvae on some selected
Poaceae plant species present in the insect’s natural habitat.;
8
3. To determine plant stimuli that influence feeding and growth of B. fusca larva:
4. To study the physiological responses / adaptations in larval digestive enzymes
and genetic structure of B. fusca population in relation to host plant use.
1. 4: Literature review
1. 4. 1: Host range and shift in phytophagous lepidoptran insects
Lepidoptera host plant affiliation is diverse with majority of larvae living at the
expense of seed plants and virtually all orders of gymnosperms, angiosperms,
ferns, liverworts and mosses. However, most clades in Lepidoptera insect family
are primarily restricted to specific higher taxa of angiosperm (Powell et al., 1998).
The diversification of Lepidoptera lineages is postulated to have paralleled or
perhaps even coevolved with their host plants (Ehrlich and Raven, 1964; Becerra
and Venable, 1999).
Most insect species use limited and specific subset of plant taxa as hosts (Strong et
al., 1984; Mitter and Farrel, 1991). Generally, within phytophagous Lepidoptera,
there is a trend that most generalists are external feeders while specialists feed
internally (Gaston et al., 1992; Frenzel and Brandl, 1998) indicating that
endophagy reduces the likelihood of oligophagy, a necessary intermediate stage
during a host shift (Drès and Mallet, 2002). Moreover, comparison between insect
specialists and generalists clearly indicate that generalists are usually fewer than
specialists suggesting that a narrow host range is favourable over evolutionary time
and space (Schoonhoven et al., 1998). A number of constraints are reported to
promote food specialisation in insects. These include, genetically based trade-offs
in performance between different habitats (for instance, habitat-specific
9
adaptations) (Via, 1991; Scheck and Gould, 1993), competition for resources
(Mac-Arthur and Levins, 1964), resistance to predators (Dyer, 1995), high cost of
information processing (Bernays and Wcislo, 1994; del Campo and Miles, 2003),
mate finding (Colwell, 1986), low costs to searching for suitable habitats
(Southwood, 1972) and habitat associated deleterious mutations (Kawecki, 1994).
In contrast, generalization is favoured by rare or unpredictable habitats (Strong et
al., 1984), difficulties with meeting nutritional requirements on a single host and
greater resource availability in terms of food supply (Singer et al, 1992; Ballenbeni
and Rahier, 2000),
Phytophagous insect species can also switch hosts and therefore expand their host
range. However, many factors including among others ecological, genetic, neuro-
physiological and/or phylogenetic conservatism potentially constraint the evolution
or maintenance of host ranges for most insect species (Jaenike, 1990; Bernays and
Wcislo, 1994; del Campo and Miles, 2003). Generally, shifts to alternative plants
are rare in monophagous insects that often feed on closely related and chemically
distinct plants. In contrast, plants in some taxonomic or ecological assemblage may
be closely related but less chemically distinct, limiting the barriers to host transfers.
This encourage insect host switch among plant species and consequently elevates
the incidence of polyphagy (Schoonhoven et al., 1998). For instance, many
herbivorous insects in temperate deciduous forests, which are dominated mainly by
tanniferous angiosperms, are highly polyphagous due to the toughness and low
nutritional content of plant mature foliage (Powell et al., 1998). Similarly insect host
diversification rates are also influenced by both resource abundance- which leads to
10
decreased competition (Fox and Lalonde, 1993; Larsson and Ekbom, 1995) and
diversity (larger number of potential niches) (Janz et al., 2006).
Several species of phytophagous insects have switched from their ancestral hosts
and rapidly adapted new plants (Singer et al., 1993; Radtkey and Singer, 1995;
Camara, 1997). However, prior to host shift insects must be able to recognize
suitable host plants based on the specific plant cues. The apple maggot fly,
Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), for example, exclusively used
hawthorn fruits until the introduction of apple in USA. However, introduction of
apple orchards and the subsequent colonisation by the fly occurred rapidly and
resulted in the formation of host races that currently differ in host choice, diapause
and alloenzyme frequency (Feder et al., 1990).
For lepidopteran species, host plant recognition is a complex process and like many
other phytophagous insects, location and subsequent selection of suitable hosts for
oviposition by the adults or feeding by larvae appears to be strongly influenced by
specific host plant search images which are based on representative chemical,
physical and visual characteristics of their host plants (Renwick and Chew, 1994;
Städler, 2002). Many examples show that typical volatile compounds emitted by
host plants guide insect herbivores while searching for hosts and therefore play an
important role in host plant recognition and selection (Honda, 1995; Bruce et al.,
2005). Similar studies on B. fusca for instance, have indicated that volatile cues
from Maize, Sorghum and Napier grass mediate host location and oviposition in
females (Khan et al., 2000; Juma, 2005; Birkett et al., 2006).
Host selection by most lepidopteran insect species is primarily a function of adult
females, since larvae in their early life stages usually have limited dispersal abilities
11
(Renwick and Chew, 1994). Therefore during host location, females maximize
larval fitness by selecting and ovipositing on plants most suitable for future larval
survival and development (Pilson and Rausher, 1988). However, as reported by
Larsson and Ekbom (1995), there is widespread ability of lepidopteran larvae to also
select and feed on hosts not selected for by gravid females. Therefore although not
highly mobile, lepidopteran larvae are not always restricted to the host plants
initially selected by their mothers. For example, for the gypsy moth, Lymantria
dispar (L.), extensive host switching can occur during larval development mainly to
improve their growth rates (Stoyenhof et al., 1994). For these larvae, switching
occurs in both high- and low-density populations indicating that other factors (for
instance host quality or phenology) dictate the dynamics of host switching. This
indicates that other than the adults, host recognition and selection behaviour by
larvae is also an important determinant of host shift (Nylin et al., 2000) and
therefore, evolutionary change in host selection may have important consequences
on host range.
1. 4. 2: Influence of plant chemistry in host choice
Phytophagous insects are able to discriminate between plants which are acceptable
for feeding and oviposition and those which are not. During host plant selection,
various types of stimuli play a role; however, chemical factors appear to be decisive
in most insect species examined (Bernays and Chapman, 1994). Host colonisations
and shifts in many species of insects have been in most cases reported to be strongly
influenced by specific phytochemicals including primary and secondary metabolites
since most of the insects studied respond behaviourally and physiologically to them.
12
For example, herbivorous insects within a defined set of hosts distribute themselves
among plants according to the specific plant secondary chemicals (Erhlich and
Raven, 1964) and may also induce physiological traits that enable them to exploit
host plants but with chemicals which in their natural form are potentially toxic to
their cellular processes (Duffey and Stout, 1996). Plant metabolites including
primary and secondary chemicals have been reported to mediate many aspects of
insect behaviour including host finding, host acceptance, danger avoidance and/or
mate location (Bernays and Chapman, 1994; Schoonhoven et al., 2005). Some of
these phytochemicals stimulate feeding and therefore act as a host plant recognition
cues. For example, larvae of Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae), a
facultative specialist when feed on solanaceous foliage usually develop preference
for indioside D, a steroidal glycoside typical of Solanaceae and hence become
habituated to such glycoside containing plants. Additionally, this host-restricted
larva is able to feed on non-host diets to which the glycoside has been added (del
Campo and Renwick, 2000; del Campo et al., 2001; del Campo and Miles, 2003),
indicating that indioside D is arecognition cue used to mediate larval host restriction.
Apart from mediating host finding and recognition, secondary plant metabolites can
also regulate larval feeding in specialist herbivorous insects and are therefore
important determinant of the suitability of a particular host plant for survival and
development (Mohamed et al., 1992). For example, the isoprenoid ketone 6, 10, 14-
trimethylpentadecan-2-one (phytone), isolated and characterised from Bermuda
grass, Cynodon dactylon (L.) (Poaceae) is phagostimulatory to larvae of the fall
armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)
(Mohamed et al., 1992). Similarly, larvae of Chilo partellus (Swinhoe)
13
(Lepidoptera: Crambidae) are stimulated to feed by soluble sorghum phenolic
compounds (4-hydroxybenzoic acid, coumaric and ferulic hydroxycinnamics)
present in sorghum ethyl acetate extracts (Torto et al., 1991). However, for majority
of oligophagous lepidopteran species, primary metabolites including sugars
especially sucrose and fructose are the most effective phagostimulants (Sharma,
1994; Bowdan, 1995; Yazawa, 1997).
Moreover, the presence of high concentration of specific phagostimulant in the host
plant tissues may in contrary elicit adverse effects to larval feeding when used over a
prolonged period of time. For instance, while soluble hydroxamic phenolic
conjugates of maize extracts for instance, 2, 4-dihydroxy-7-methoxy-1, 4-
benzoxazin-3-one (DIMBOA) usually stimulate feeding of European corn borer
(ECB) Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae), long-term feeding of
DIMBOA throughout larval development, usually results in reduced fecundity and
relative growth rates (prolonged development) of ECB (Bergvinson, 1993) or
Ostrinia furnacalis (Guenee) (Ortego et al., 1998).
However, the phagostimulatory and inhibitory effects of plant chemicals, including
primary and secondary compounds, often counteract each other and determine the
outcome of insect’s decision making process, to accept or reject the plant as a host.
In such cases, if all the sensory information gathered during larval test bite is
positively judged, along with any physical cues offered by the plant, acceptance of
the host is confirmed and feeding is initiated (Schoonhoven et al., 1998). The effect
of phytochemicals on insect ability to choose a host therefore clearly indicates that,
chemical diversity among plants is an important factor underlying host specificity in
phytopagous insects.
14
1. 4. 3: Phylogenetic constraints to host use
Generally, phytophagous insects utilize diverse food resources over their entire
geographical range though localized populations may experience different selection
pressures. Populations from a distinct region can adapt and utilize a specific host
such that a putatively generalist species may actually consist of specialist local
populations. This implies that oligophagy may not necessarily mean oligophagy at
the individual level (Thompson, 1994). Many clades of herbivorous insects are
remarkably conservative in the plants they attack and in many groups; related
insects tend to feed on related plants (Erhlich and Raven, 1964; Janz and Nylin,
1998). Hence, species in a higher insect taxon such as the genus or subfamily, will
commonly feed on taxonomically related plants, often of the same family. This
conservative feeding habit strongly reflects the existence of constraints that lower
the ability of insects to adapt to plants that are distantly related to their normal hosts
(Schoonhoven et al., 1998).
Insects colonize plants that produce specific chemicals and thus plant chemistry
plays an important role in determining the variety of plants that can be exploited by
phytophagous insects (Bernays and Chapman, 1994). Therefore, shifts to and the
subsequent colonization of alternative plants by related groups of phytophagous
insects often arise as a result of tracking of specific and suitable plant phytochemical
cues in the novel host (Beccera, 1997; Nylin and Janz, 1999). In most cases, such
insects become physiologically pre-adapted to a new host plant’s secondary
compounds that are often not similar to those of the ancestral host. For example, the
association between various insects and plants containing furanocoumarins have
been interpreted according to this scenario (Camara, 1997). Similarly, related
15
species of butterflies and beetles often use plants that are chemically similar, even
though these plants may be taxonomically distant (Ehrlich and Raven, 1964; Janz
and Nylin, 1998). Similarly, although cladograms of papilionid butterflies and
chrysomelid leaf beetles (genus Ophraella) and their host plants are not congruent
(Jaenike, 1990), their cladistic analyses reveal that host shifts in these group of
insects are most likely to occur among chemically similar plants.
A residual capacity by insects to use ancestral hosts not in the present range, long
after the colonisation of the novel host plant has been described for some insect
species and can be evident in a clade many million years following the colonization
of a novel host (Janz, et al., 2001; Ikonen et al., 2003). The fact that these ancestral
hosts are often kept in a potential repertoire long after colonisations of novel hosts,
and frequently re-colonised at a later date, suggests that the observed insect
diversification is not due to release from competition but may be due to greater
niche diversity as a consequence of greater plasticity in taxa with wider potential
host range (Nylin and Wahlberg, 2008).
On the other hand, phylogenetic constraint to host shift is not universal among
insects since similar host plant shifts by insects have been documented among plants
that are distantly related but chemically or structurally different from their ancestral
host species (Dobbler et al., 1996; Crespi and Sandoval, 2000). Species of the leaf
beetle of the genus Tricholochmaea specialise on willow (Salicaceae), blueberry
(Ericaceae), or Meadowsweet (Rosaceae), which tend to grow in similar habitats but
do not otherwise have common physical and chemical traits (Futuyma, 1999). Shifts
to taxonomically unrelated plants is however, dependent on the geographical
distribution of hosts and thus availability of plants (i.e., ecological opportunity),
16
insect learning or adaptive plasticity among many other constraints (Gomez-Zurita
et al., 2000).
Variations in food choice and utilisation among insect populations may also be
caused by insect genetic differentiation as a result of differences in preference
induction. Induced preference can initially occur as a result of oviposition mistakes
by the adult females (Larsson and Ekbom, 1995; Nylin et al., 2000). If such and
similar mistakes occur often enough and persist over several generations and keep
inducing the modified phenotype, then genetic changes that improve fitness to this
specific host are likely to be selected as a result of genetic accommodation (Janz and
Nylin, 2008). Similarly, induced preference can also minimise host fidelity and
decrease movement to alternative hosts consequently leading to genetic sub-
structuring within species (Via, 1991). If gene flow between such populations is
limited, this sometimes results in the formation of host-associated sub-populations
or host races (Kim and McPheron, 1993; Berlocher and Feder, 2002). Host races are
populations of a species partially and reproductively isolated from the other
conspecific populations as a direct consequence of adaptations to a specific host.
Similarly, speciation can also occur when a specialist’s host is patchily distributed
increasing the likelihood of differentiation among populations by restricting gene
flow. In many cases therefore, diversification to alternative host plants by many
insects species are often phylogenetically constrained to taxonomically, chemically,
or structurally similar host species (Futuyma and Moreno, 1988; Janz and Nylin,
1998; Janz et al., 2001).
17
1. 4. 4: Physiological constraints to host use
The physiological basis of selective feeding in phytophagous insects has been
examined in many studies, and in most cases, host plant discrimination depends
largely on the chemical content of the food plant. Physiologically, phytophagous
insects usually possess traits that enable them to exploit chemically diverse host
plants, since most of them must deal with toxic or non-nutritive plant secondary
chemicals potentially damaging to their cellular processes (Duffey and Stout, 1996).
Berenbaum (1990) postulated that specialist insects possess specific enzyme systems
capable enough of degrading potential toxins predictably encountered. The
production of these enzymes in many insect species, results from mutation of a gene
that codes for enzymes that allow the detoxification of the ingested compounds prior
to their sequestration in some target organs. If such mutations are advantageous,
high gene-flow occurs rapidly to spread the new allele to the whole population and
allow adaptations to a new host plant.
Detoxification of plant allelochemicals by specialised insect enzyme systems is
widely believed to be the most important factor contributing to the adaptations of
insects to different plant diets, host shift and subsequent diversity in host range. For
example, the gut microsomal mixed function oxidases (MFO) have been widely
implicated in the detoxification of toxic plant allelochemicals through oxidative
reactions in many insect species (Brattsten, 1988; Feyereisen, 1999) and hence play
an important role in host range determination.
However, although cases of trade-offs in physiological adaptations to different host
plants have been described for some insect species and proposed to underlie
speciation or the evolution of speciation in phytophagous insects, other factors
18
including neural constraints to host recognition (Bernays, 2001) or possibly,
physiological trade-offs in adaptation to abiotic conditions are often likely to have a
greater effect on host plant choice in most insects.
1. 4. 5: “Learning” as a mechanism of host switch
Host switches in insects can also occur without constraint from lineage or chemistry
and in most cases may be mediated by learning or induced preference (Tallamy,
2000). Jermy et al. (1968) defined induction as the modification of feeding
behaviour by a change in host plant preference due to the previous feeding
experience. A shift to taxonomically or chemically unrelated novel hosts is primarily
mediated by induced preference or adaptive plasticity by insects due to experience
(Huang and Renwick, 1995; Agrawal et al., 2002). For instance, previous
experience has been shown to have a substantial effects on the rate at which some
butterflies locate particular host plants (Papaj, 1986).
In phytophagous larvae, induced feeding preference is an outcome potentially
caused by a number of behavioural and physiological mechanisms including
habituation to deterrents, associative learning and sensitisation, ‘oviposition
mistakes’ or host deprivation (Huang and Renwick, 1995). The mechanisms by
which sensitivity is induced or suppressed in young larvae are not yet known but
possibly after hatching, the peripheral gustatory receptor of neonate larvae may
become quite malleable and permanently moulded in ways that affect the
acceptability of leaf tissue as food source. Conversely, the presence of one or more
deterrents in a novel host can permanently suppress the development of sensitivity
to these and other compounds thus enabling neonate larvae to feed without ill
19
effects. This implies that neonate larvae are far more plastic in their food acceptance
criteria than the old larvae. However, this is only possible if there is no prior
exposure of the larvae to the food that lacks a particular deterrent (Tallamy, 2000).
Induction of host preferences for both larval and adult insects has been examined in
different insect orders such as Lepidoptera (Anderson et al., 1995; Huang and
Renwick 1997; Akhtar and Isman, 2003), Diptera (Jaenike, 1988) and Coleoptera
(Rietdorf and Steidle, 2002). In most of these cases, previous exposure to deterrent
compounds significantly reduced aversion at subsequent encounters. Dietary
experience can influence the ability of insects to taste plant chemicals that signal the
feeding suitability of the host. Therefore, repeated exposure of herbivorous insects
to a specific non stimulus plant or diet can lead to an increased preference of the
host species (Hopkins, 1917) and possibly lead to host switch.
Although, induction of food preference within the larval stages has been reported for
many insect groups, only few investigations indicate that larval experience alters
adult preference or acceptance to hosts (Anderson et al., 1995; Akhtar and Isman,
2003). Moreover, prior adult females encounter or oviposition on a given host is also
necessary for an increased likelihood of future insect preference or acceptance of the
specific host plant (Papaj, 1986; Cunningham et al., 1998; Egas and Sabelis, 2001).
For most lepidopteran species, it has been demonstrated that early larval experience
is normally transmitted through metamorphosis and this may explain conservatism
of plant preference observed in majority of Lepidoptera species (Barron, 2001;
Blackiston et al., 2008). Modification of preference by experience either during
adulthood or larval stage is however, not universal (Parmesan et al., 1995).
20
1. 4. 6: Ecological constraints to host choice
Phytophagous insect diversification rates can conceivably be influenced by
geographic variations in the relative abundance of resource leading to decreased
competition among insects utilising the same food source or due to diversity in
plants (larger numbers of potential niches) (Singer et al., 1992; Denno et al., 1995).
For instance, in areas where top-ranked hosts are abundant and oviposition frequent,
thresholds for host acceptance would remain high, precluding use of low ranking
hosts. However, where favoured plants are rare or their presence is masked by
associated members of the plant community, thresholds for host acceptance
decrease, making the use of other plants more likely subsequently leading to host
range diversification (Singer et al., 1989). Resource diversity therefore creates
opportunities for insects to shift to novel host plants (Nylin and Wallberg, 2008).
Depending on the spatial arrangement of plants, the scale of intercrops and the
movement of insects, intercropping may lead to repeated encounter of non-host
plants, greatly contributing to oviposition on non-host plants partly due to
oviposition mistakes and subsequent host switch.
1. 4. 7: Influence of plant physical attributes to host choice
Plant selection and acceptance by phytophagous insects is also determined by plant
physical characteristics (Calatayud et al., 2006). Prior to feeding on the plant
tissues, insect larvae generally palpate the plant surface as a direct response to plant
contact cues. Upon contact, larvae obtain information on plant quality for which
mechano-chemosensory stimuli are involved. Plant physical factors including the
presence of trichomes and wax crystal structures, leaf thickness and toughness,
21
sclerotization and high silica content on the plant surface may strongly influence
the avoidance feeding behaviour of larvae (Shoonhoven et al. 1998).
Among the possible plant physical attributes, silica (Si) is among the common
elemental chemicals that can accumulate in plant tissues (Jarvis and Jones, 1987;
Sangster et al., 2001) and determine host choice in phytophagous insects. The
protective effect of silica to plants against insect herbivores, pathogens or abiotic
factors is related to the level of its accumulation and polymerization in the plant
tissues with highest levels positively correlating with highest resistance to insect
feeding (Meyer and Keeping, 2005; Laing et al., 2006). For lepidopteran species,
mitigating effects of silica against borer attack has been observed in barley, rice
wheat, sugarcane, maize and sorghum (Schoonhoven et al., 2005; Kvedaras et al.,
2007a).
Although mechanisms of silica mediated resistance to insect herbivore is still scanty,
(Ma, 2004; Hammerschmidt, 2005), silica in plant epidermal cells is thought to
provide a physical barrier against borer probing and feeding or pathogen penetration
into plant tissues (Djamin and Pathak, 1967; Peterson et al., 1988; Kvedaras and
Keeping, 2007). Silica may also increase leaf abrasion which subsequently increases
wearing of insects’ mandibles and therefore physically deter larval feeding (Raupp,
1985; Massey et al., 2006). Biochemically, silica has been reported to modulate the
accumulation of herbivore defensive allelochemicals including phytoalexins, lignin
and phenolics in plant tissues (Cherif et al., 1994; Rodrigues et al., 2004; Remus-
Borel et al., 2005) or induce the production of plant defensive enzymes such as
peroxidase, polyphenoloxidase and phenylalanine ammonia lyase in response to
herbivorous insect attack (Keeping and Meyer, 2002; Gomes et al., 2005). These
22
defensive plant enzymes take part in a number of plant defense processes such as
lignifications and/or production of antiherbivore plant metabolites (Goodman et al.,
1986; Felton et al., 1994). Nevertheless, the effects of plant tissue silification as a
defense mechanism against insect herbivores seem not universal. For example, high
silica levels in turfgrass had no influence on feeding and development of
Herpetogramma phaeopteralis Guenée (Lepidoptera: Pyralidae), nor on growth,
survival, feeding preference or mandibular wear of Agrotis ipsilon (Hufnagel)
(Lepidoptera: Noctuidae) (Redmond and Potter, 2007).
1. 4. 8: Neural constraints to food choice
Constraints on information processing might help to explain the general tendency
of herbivorous insects to specialise on relatively few host plants (Fox and Lalonde,
1993; Bernays and Wcislo 1994; Larsson and Ekbom, 1995). Dusenbery, (1992)
predicted that identifying, discriminating and choosing among potential host plants
usually present a challenge to most phytophagous insects because insect nervous
systems have limited capacity to process multiple sensory inputs. The capabilities
of insects to discriminate among plant species and the ways in which they use the
available information to make such decisions often have profound effect in diet
breadth beyond host quality (Bernays and Chapman, 1994; Bernays 2001) and
depend on the sensory information gathered during taste bite. When such
information is judged positively by the central nervous system, acceptance, the
final decision taken in the host-plant selection process, is confirmed and food
intake occurs. From an evolutionary perspective, acceptance can then be
23
considered as the crucial decision taken during host-plant selection, as it has direct
consequences for the acquisition of nutrients (Schoonhoven et al., 1998).
Lepidopterous larvae have four sets of external chemosensory organs: the
antennae, the maxillary palps, the medial and lateral maxillary sensilla styloconica
and the epipharyngeal sensilla (Schoonhoven, 1987) that are used in food plant
choice. Stimulation of the antennal olfactory receptors by plant odours usually
evoke short range orientation towards a plant, whereas activation of the olfactory
cells associated with the maxillary palpi may have a phagostimulatory effect
(Ishikawa et al., 1969). Gustatory receptors present in the palpi, the maxillary
sensilla styloconica and the preoral and buccal cavity (Ma, 1976), further
determine acceptability of the plant. Therefore, host-restricted larvae often choose
their food based on input from taste receptor cells located within chemosensory
sensilla on their mouthparts. Antennal, maxillary palp and epipharyngeal sensilla
for example, have been implicated to respond to chemical cues of host plants in
non host restricted Manduca sexta larvae but seem to have less significant role in
the feeding behaviour of host restricted Solanaceae feeding larvae (Glendinning et
al., 1998). The responses of the sensilla styloconica to a variety of chemicals
compounds have also been examined in a number of insect species. For M. sexta,
feeding is mediated entirely by the sensilla styloconica on the galea, as removal of
these sensilla completely eliminates food preferences of the host-restricted larvae
(Flowers and Yamamoto, 1992; del Campo and Miles, 2003). This means that the
sensory input from sensilla is both necessary and sufficient for host recognition by
host restricted larvae.
24
As a result of neural confusion, ovipositing insects may ignore the more suitable
food for their larvae possibly because host plants lack required recognition cues,
present deterrent but harmless chemicals or conversely, may accept unsuitable host
plants because of presence of oviposition stimulants (Fox and Lalonde, 1993). On
such occasions insect will ultimately select a host plant not within the host range.
Another approach in the search for the chemosensory mechanisms involved in food
choices involves studying the neural responses of larval chemosensory organs to
deterrent compounds. When such compounds are added to artificial diets they
modify taste sensilla responses by habituation, resulting in decreased sensitivity to
the available stimulus (van Loon, 1990; Glendinning and Gonzalez, 1995).
Knowledge of preference behaviour requires a study of the functions of the
chemoreceptors involved and the electrophysiology offers a relatively rapid and
precise method to elucidate the chemical factors by which plant is recognized.
Comparisons of the electrical responses of the chemoreceptors on stimulation with
different substances may reveal which chemicals the insect selects through its sense
organs from the complex chemical environment.
For B. fusca larvae, the chemosensory basis of food plant selection is still at infancy
and a more comprehensive study investigating the role of all the known
chemoreceptors and important in the discrimination of host from the non-host plants
is needed.
25
CHAPTER TWO
2.0: DISTRIBUTION OF CHEMO AND MECHANORECEPTORS
ON THE ANTENNAE AND MAXILLAE OF B. FUSCA LARVAE
2. 1: Introduction
Phytophagous insects lay their eggs on plants based on the preferential selection of
their host plants. Host finding and acceptance foraging behaviour for such insects
are often constrained to particular host species that are thought to represent the
suitability of hosts for future larval survival. Host recognition and acceptance in
many phytophagous insect species are usually governed by the adults although host
recognition and acceptance by larvae is widespread and equally important
(Roessingh et al., 2007).
Like many other Lepidoptera, the success of B. fusca to recognize and colonize a
limited variety of plants is based on the interactive effects of its sensory system and
the physico-chemical characteristics of the immediate environment (Calatayud et al.,
2006). After hatching under the leaf sheath, B. fusca neonates initially ascend to the
leaf whorl, where they either feed on the leaves or disperse to other plants via
‘ballooning-off’ effect (Kaufmann, 1983). However, upon feeding on the leaf whorls
and gaining appropriate size, third instar larvae generally descend and bore into the
plant stem or migrate in search for more suitable feeding part of the host plant
(Kaufmann, 1983). This indicates striking food selective responses by B. fusca larva,
which appears to be mediated by neural tuning and influenced by larval age and
specific plant tissue.
26
Selective feeding preferences displayed by lepidopteran larvae are based on small
set of chemoreceptors present on the larval antennae and maxilla and are thus
important in host plant recognition and selection (Roessingh et al., 2007). It is
however, not clear whether the sensory structures and their inherent sensitivity to
various plant stimuli change with larval age so as to enable the detection of cues
emanating from different parts of the host plants used as food source.
Therefore, as a first step to elucidate the basis of host plant recognition and
selection, the external morphology and distribution pattern of sensilla present on the
maxillae and antennae of neonates and third instar of B. fusca larvae were studied.
The olfactory and / or contact chemosensory functions of these sensilla were
identified based on direct observation made using selective silver staining and dose-
response electrophysiological tests.
2. 2: Materials and Methods
2. 2. 1: Insect rearing
Neonates and third instar B. fusca larvae were obtained from the Animal Rearing
and Containment Unit (ARCU) of the International Centre of Insect Physiology and
Ecology (ICIPE, Nairobi, Kenya). Neonates were directly used for experiments one
day after hatching. To obtain third instar larva, some neonates were reared on a
meridic diet as described by Onyango and Ochieng’-Odero, (1994). Briefly, boiled
and cooled (60°C) distilled water was mixed with preweighed quantity of
ingredients as listed (in Table 2.1) to make fraction A of the diet. The mixture was
then blended in warring blender for three minutes. Fraction B of the diet was made
by mixing cold distilled water with a preweighed agar powder and boiling before
27
cooling to 60°C. Ingredients of fractions A and B were then mixed and blended
together for further 3 minutes. 40% of formaldehyde (fraction C of the diet) was
measured and mixed with ingredients of fraction A and B and the mixture once
again blended for further 3 minutes. Approximately 20 ml of the resulting diet was
dispensed into each heat-sterilised glass vial (7.5 cm long x 2.5 cm diameter) using a
ketchup dispenser. The diet containing vials were then covered with a clean white
cloth and left overnight on the bench in the laboratory to cool and gel.
Four, one day old neonate larvae were inoculated in each vial containing cooled
artificial diet. The vials were then stoppered using a cotton wool and larvae allowed
to feed and grow in a temperature controlled room until third stadium (after 21-24
days) before they were used for bioassay experiments.
Table 2.1: Amount of ingredients used to prepare a litre of artificial diet used to rear
Busseola fusca larvae (Onyango and Ochieng’-Odero, 1994)
Ingredients Quantity (g) per litre diet Fraction A : Distilled water
404 ml
Bean powder 88.4 g Maize leaf powder 25.4 g Brewers yeast 22.7 g Ascorbic acid 2.5 g Vitamin E 2.1 g Sorbic acid 1.4 g Methyl p-hydroxy benzoate 2.0 g Sucrose 35.4 g Fraction B : Agar Techno. N°3
12.6 g
Distilled water 404 ml Fraction C: Formaldehyde 40%v/v
2 ml
28
2. 2. 2: Scanning electron microscopy
Ten live neonates and ten decapitated heads of third instar larvae were used for the
preparations of specimens for scanning electron microscopy. For fixation, the
specimen were allowed to stay overnight in a 2.5 % glutaraldehyde prepared in 0.1
M phosphate buffer (pH 7.4) solution. The specimen were then dehydrated in a
graded series of ethanol (70%, 90% and 100%) and finally air-dried. The heads of
neonates were then separated from the rest of the bodies. Larval heads for both
insect developmental stages were then mounted on stubs with conductive double-
side adhesive tape, sputter-coated with gold, and finally examined with a JEOL
JSM-T330A scanning electron microscope at 10 kV.
2. 2. 3: Silver nitrate staining
Silver nitrate staining was done to determine the presence of pores in the maxillary
and antennal sensilla on the heads of neonates and third instars. Intact neonate larvae
were stained according to the method of Nayak and Singh (1983) modified as
follows: Larvae were first immersed in 70% ethanol containing 1 M silver nitrate for
1 hour and then dehydrated in two concentrations (90% and 100%) of ethanol.
Afterwards, heads of neonates were separated from the rest of insect’s body. Heads
of both instars were separately cleaned in xylene overnight. The specimens were
then mounted in Mountex (Histolab) for light microscope observations. A total of 10
head specimens from each of larval developmental stages were examined for pores
and distribution of the sensilla.
29
2. 2. 4: Electrophysiology
Electrophysiological tip recordings from sensilla on the maxillae and antennae of
third instar larvae were carried out to determine the contact chemosensory function
of the sensilla using tip recording technique (Hodgson et al., 1955). Recordings
were carried out only for third instar larvae because the sensilla present on neonates
were too small to make electrical contact possible. Larvae were first severed at the
thorax and a glass micropipette (recording electrode-thin walled borosilicate glass
capillaries, Harvard apparatus) filled with 10 mM NaCl inserted into the body part
that contained the head. The glass micropipette was then slipped over a silver wire
that served as the reference electrode. The sensilla were then probed for electrical
contact with another micropipette filled with 100 mM KCl that sheathed another
silver wire and grounded to act as the indifferent electrode. To avoid crystallization
and concentration changes at the tip, the electrode was filled with the test substance
just a few seconds before the start of the recording. 10 individual larval parts were
probed for electrical contact. The recording electrode containing the test solution
was placed over single sensilla hairs for 5s with an interstimulus interval of
approximately 10 minutes to avoid adaptation. The action potential generated were
amplified using a universal AC/DC UN-06 amplifier (Syntech, The Netherlands),
recorded on a computer and analyzed using Autospike software (Syntech version
2.1a). The analyses were based on the waveform and amplitude properties of the
action potentials generated. A dose-response experiment using sucrose, a feeding
stimulus for B. fusca larvae when reared on artificial diets (Onyango and Ochieng’-
Odero, 1994), was done for sensilla that showed action potentials to provide
evidence for a contact chemosensory function. Sucrose concentrations of 0.01, 0.1,
30
1, 10, 100 and 500 mM prepared in 10 mM KCl were used. The nervous impulses
generated by different neurones in the sensilla were discriminated using Autospike
software (Syntech version 2.1a).
2. 2. 5: Data analysis
Statistical tests were performed with Statview software (Abacus Concept, version
5.0, USA). For data on the sensilla lengths, means were separated by Mann-Whitney
U-test. The spike counts during the first 150 ms of each recordings were log(x+1)
transformed in case of electrophysiological data. A linear-regression of log (number
of spikes +1) on log (concentration) was done and tested using ANOVA.
2. 3: Results
2. 3. 1: Sensilla present on the larval antennae
Scanning electron microscopy data indicated that the antennae of both neonates and
third instars larva of B. fusca comprise of three segments (Figure 2.1 a). Located on
these segments are three typical insect sensilla: sensilla chaetica, sensilla styloconica
and the basiconic sensilla. On the second antennal segment are a pair and dorsally
positioned aporous sensilla chaetica (Figure 2.1b, marked C1& C2) and two cone-
shaped basiconic sensilla (Figure 2.1b, marked 1 & 3). A similar cone-shaped
basiconic sensillum is located on the third antennal segment (marked as 2 in Figure
2.1b). The three cone-shaped basiconic sensilla on the second and third segments are
of equal lengths and were shown to possess argyrophilic properties (Figure 2.1c)
since they allowed silver nitrate dye to penetrate through their pores and precipitate
in their lumen. Three additional but small basiconic sensilla are also present on the
31
antennae; one located on the second segment while the other two on the third
antennal segment (Figure 2.1b, marked as 1’, 2’, 3’). The presence of pores on the
small basiconic sensilla was not discernable and hence their function could not be
clearly determined in this study. One aporous styloconic sensillum is present on the
third antennal segment adjacent to the cone- shaped basiconic sensillum. Similar
type and number of sensilla but with variable sizes were found on the antennae of
both neonates and third instar larvae. The size of these sensilla positively correlated
with larval developmental stage. For each instar, sensilla chaetica was the longest,
then the large basiconic sensilla and finally sensilla styloconicum (Table 2.2).
The action potential obtained for the cone-shaped basiconic sensillum located on the
third antennal segment (marked 2 on Figures 2.1b and c) following
electrophysiological tip recordings indicated a gustatory function (Figure 2.2).
However, similar sensilla on the second segment and the other small basiconic
sensilla present on both second and third antennal segments (1 and 3 on Figures 2.1b
and c) did not show any action potential activity during the tip recording tests.
Table 2.2: Meana lengths (µm, ± SE; n=4) of antennal sensilla of Busseola fusca
neonate and third instar larvae
Sensilla type Larval stage
Long sensillum
chaeticum (C1)
Short sensillum
chaotic (C2)
Large anionic
sensilla (1, 2, 3)
Styloconic
sensillum (S)
Neonates 47.4 ± 3.7 a 5.7 ± 0.4 a 8.6 ± 0.9 a 7.6 ± 0.5 a
Third instar 200.7 ± 31.3 b 35.2 ± 2.7 b 25.4 ± 2.9 b 14.8 ± 1.1 b aMeans followed by different letters are significantly different at 5% level
(Mann-Whitney U test).
32
Figure 2.1: Antenna of third instar Busseola fusca larva. (A) Tip of right antenna
showing the dorsal view of segments I to III. (B) Details of figure A showing two
aporous sensilla chaetica (C1, C2), dorsally positioned on the second segment; three
cone-shaped basiconic sensilla of similar lengths, two located on the second
segment and one on the third segment (1, 2, 3); three small basiconic sensilla (1’, 2’,
3’) and one aporous styloconicum sensillum (S) on the third antennal segment. (C)
Dorsal view of the left antenna tip showing 3 large silver stained basiconica sensilla
on the 2nd and 3rd segments following staining with silver nitrate.
A B
C
33
Figure 2.2: Electrophysiological spike activity recordings of the basiconic sensillum
on the third antennal segment of a third instar larva in response to 100 mM KCl.
Vertical bar: 10 mV, horizontal bar: 200 ms.
Figure 2.3: Dose-response curves for sucrose obtained after contact with the cone-
shaped basiconic sensillum on the third antennal segment (A) and with the maxillary
palp (B) of third instar larva. The numbers of replicates are given in parenthesis.
Error bars indicate standard error.
200 ms
B A
34
2. 3. 2: Sensilla present on the larval maxillae
The sensilla on the maxillary galeae of B. fusca larvae were found to comprise of
two styloconic sensilla (MS, LS), three basiconic sensilla (B1, B2, B3), and two
sensilla chaetica (C1, C2) (Figure 2.4a). The lateral and medial styloconic sensilla
stained with silver nitrate and hence were shown to contain a single terminal pore
(uniporous) (Figure2.4b). Electrophysiological tip recordings of the neuronal
activity obtained from lateral and medial styloconic sensilla (Figures 2.5a and b)
indicated their gustatory function. Three aporous basiconic sensilla are located
dorsally to the styloconic sensilla (Figure2.4a) while two aporous sensilla chaetica
are situated dorsally on the distal part of each galea (Figure 2.4a).
The maxillary palp is two-segmented and contains 8 small basiconic sensilla at the
tip (Figure 2.4a) for both neonates and third instar larvae. The number of pores on
each small basiconic sensilla on the maxillary palp was not visible but readily
stained with silver nitrate indicating their porous property (Figure 2.4c). Similarly,
electrophysiological tip recording on each individual basiconic sensilla at the tip of
the maxillary palp was impossible because of sensilla’s minute size. However,
electrical contact and spike strains recorded from this tip (Figure 2.5c) indicated a
gustatory function of the palp. As for the antennal sensilla, both larval
developmental stages consisted of same types and equal number of sensilla though
significantly variable in size. Sensillae chaetica were the longest while styloconic
sensilla were the shortest for both larval developmental stages (Table 2.3).
A dose-response electrophysiological tip-recording experiment using sucrose was
done on the sensilla present at the tip of the larval maxillary palp and on the cone-
shaped basiconic sensillum present on the third antennal segment in order to confirm
35
their contact chemosensory function. The tip of the maxillary palp was chosen for
two reasons: i) it harbours both multiporous and/or uniporous sensilla that have
spike trains generated by contact electrophysiological recording (Figure 2.5c) and ii)
it is known to respond positively to sucrose in lepidopteran larvae (e.g., Albert,
2003). In this study, significant positive dose-response curves were obtained for
both the antennal sensillum (F1, 56 = 41.637, P<0.0001; Figure 2.3a) and the
maxillary palp (F1, 58 = 32.124, P<0.0001; Figure2.3b) when the concentration of
sucrose was increased.
Table 2.3: Meana lengths (µm, ± SE; n=4) of maxillae sensilla of neonate and third
instar larvae of Busseola fusca
Sensilla type
Larval stage Long sensillum
chaeticum (C1)
Short sensillum
chaeticum (C2)
Styloconic
sensilla (LS, MS)
Maxillary
palp (P)
Neonates 24.5 ± 0.6 a 12.9 ± 1.3 a 3.7 ± 0.05 a 12.1 ± 0.9 a
Third instar 113.4 ± 0.5 b 43.6 ± 4.4 b 6.1 ± 0.6 b 28.0 ± 3.5 b aMeans followed by different letters are significantly different at 5%
level (Mann-Whitney U test).
36
Figure 2.4: Maxilla of third instar Busseola fusca larva. (A) Left maxillary galea
showing uniporous lateral and medial sensilla styloconica (LS, MS), 3 sensilla
basiconica (B1, B2, B3), 2 sensilla chaetica (C1, C2) and the left maxillary palp (P)
with 8 sensilla basiconica on the tip and showing laterally a sensillum digit form
(see arrow). (B) Close-up of the uniporous lateral and medial sensilla styloconica
(LS, MS) with a terminal pore on each. (C) Tip of the right maxillary palp (P), seen
dorsally and stained following silver nitrate staining procedure.
C1
P
C2
B1
B2
B3 LS
MS
50 µm
A
B C
37
Figure 2.5: Contact electrophysiological recording of spike activity after contact
with a lateral sensillum styloconicum on the maxillary galea of a third instar larva
(A), with a medial sensillum styloconicum on the maxillary galea of a third instar
larva (B) and with a maxillary palp of a third instar larva (C) in response to 100 mM
KCl. Vertical bar: 20 mV, horizontal bar: 200 ms.
A
B
C
200 ms
38
2. 4: Discussion
The antennae of B. fusca larvae are short and simple and comprise of only three
segments. The distribution, number and types of sensilla on neonates and third instar
larvae are similar and their structure parallels results for other larvae of lepidopteran
families, including the Noctuidae, Sphingidae, Arctiidea and Lasiocampidae
(Dethier, 1937; Faucheux, 1999). However, this contrasts results obtained for the
sensilla present on the heads of the adults of B. fusca moths which are long and
complex (Calatayud et al., 2006). On the second antennal segment, two sensilla
chaetica are dorsally positioned. Unlike for adult moths whose sensilla chaetica
were porous, similar larval sensilla chaetica were aporous as was indicated by the
non-penetration of silver dye in their lumen. As suggested by Dethier (1941) and
Faucheux (1999), these aporous sensilla may be adapted for detection of tactile
stimuli, thus, informing the insect of obstacles in its immediate environment and of
the contours on the feeding substrate.
Three cone-shaped basiconic sensilla of approximately similar lengths are also
present on B. fusca larval antennae. The walls of basiconic sensilla are generally
perforated by numerous pores (Faucheux, 1995; 1999) indicating that the sensilla
may present an olfactory function. Olfactory sensilla have receptor neurons that play
an important role in discriminating odours that emanate from different plants
(Dethier and Schoonhoven, 1969; Dethier, 1980; Faucheux, 1999) and hence are
important in the location and selection of suitable hosts. Preliminary results have
indicated that both neonates and third instar larvae of B. fusca orient significantly
towards maize plant volatiles in Y-tube olfactometric tests. Moreover, the
electrophysiological tip recordings on one basiconic sensillum located on the third
39
antennal segment indicated that it has a gustatory function as well. Preliminary
observations of the behaviour of second instars showed that prior to feeding; larvae
generally tap the plant leaf surface with the tip of the maxillary palp, suggesting
evaluation of the plant surface (Calatayud et al., 2006). Furthermore, the cone-
shaped basiconic sensilla located on the third antennal segment seem to frequently
touch the plant surface during this tapping behaviour, indicating a probable
involvement of both sensilla in plant surface evaluation. However, this plant tapping
behaviour with antennal sensilla has not been clearly observed and additional studies
are necessary for confirmation in order to elucidate the actual role of these sensilla
in plant surface evaluation. Although, the presence of pores on the small basiconic
sensilla present on the third antennal segment could not be clearly determined in this
study, these sensilla are generally multiporous and are probably thought to have
olfactory receptor neurons (Faucheux, 1999). In addition, the third antennal segment
has one aporous styloconic sensillum. This sensillum may be innervated by cold
sensitive receptors (Schoonhoven, 1967; Faucheux, 1999).
Similar to several other Lepidoptera families (Faucheux, 1999), the sensilla on the
maxillary galeae of both neonates and third instar B. fusca larvae comprise of two
uniporous styloconic sensilla (lateral and medial), three aporous basiconic sensilla
and two aporous sensilla chaetica. These sensilla showed a marked variation in size
between the two larval developmental stages. Electrophysiological tip recordings of
the neuronal activity obtained from the uniporous styloconic sensilla on the
maxillary galeae confirmed their gustatory function, as has been reported for several
other larvae of lepidopteran species (Ishikawa, 1963; Schoonhoven and Dethier,
1966; Dethier and Kuch, 1971; Faucheux, 1999). For all lepidopteran larvae that
40
have been studied, the styloconic sensilla respond to many different plant
compounds and therefore play a major role in discriminating among plant
constituents during insect’s initial biting process and sustained feeding (Faucheux,
1999). It is therefore proposed that host plant discriminatory ability of B. fusca
larvae may accrue from the differential responses of the chemosensory neurones in
the sensilla stylonica and forms the basis of selective food choices by this
oligophagous B. fusca larva.
The aporous basiconic sensilla, located dorsally of the styloconic sensilla, may play
a role in thermo-hygroreception and in proprioreceptive function by monitoring the
position of the styloconic sensilla (Shields, 1994; Faucheux, 1995; 1999). Moreover,
the two aporous sensilla chaetica, situated dorsally on the distal part of each galea,
probably have proprioreceptors that respond to mandibular movements during
feeding as reported in similar studies (Grimes and Neunzig, 1986; Faucheux, 1999).
Three possibilities are reported for other larvae of lepidopteran species on the
function of the small basiconic sensilla present at the tip of the maxillary palp. First,
all the 8 may be uniporous basiconic sensilla (the most frequent case). Secondly,
they may occur as a combination of 7 uniporous basiconic sensilla and one
uniporous styloconic sensillum [e.g., in Choristoneura fumiferana (Clemens)
(Lepidoptera: Tortricidae)]. Thirdly, they may consist of 5 uniporous and 3
multiporous basiconic sensilla [e.g., in Pieris brassicae (L.) (Lepidoptera: Pieridae)
and Manduca sexta L. (Lepidoptera: Sphingidae)] (Faucheux, 1999). Although it
was not possible to carry out electrophysiological tip recording tests on each
individual sensilla, the electrical contact and spike strains recorded from the overall
tip of the maxillary palp confirmed the gustatory function of the sensilla on the tip of
41
the maxillary palp as has been reported for other lepidopteran larvae (Devitt and
Smith, 1982; Faucheux, 1999; Albert, 2003).
In conclusion, B. fusca larvae have sensory structures that are able to detect volatiles
and surface chemical stimuli on their host plants. The olfactory receptors are mainly
located on the antennae but they are also present on the maxillary palps. Gustatory
chemoreceptors occur mainly on the maxillae and their presence on the antennae of
B. fusca larvae was demonstrated for the first time and the presence of similar
sensilla has never been reported before for any other lepidopteran larvae. This is
particularly interesting in view of the antennae and the maxillary palps having
receptors that are important in mediating food selection for example, as has been
reported in M. sexta (De Boer, 1993). The results presented in this study show that
the antennae indeed has a chemosensory function, and might be involved in the
assessment of host plant suitability. The presence of gustatory sensilla on the
antennae offers the larvae with an enhanced ability to quickly and efficiently
evaluate appropriate phagostimulatory non-volatile chemical cues present on the
plant surface following landing on the plant surface prior to the adoption of plant as
a host. This may partly explain the ability of B. fusca larvae to easily include a
number of host plants with phagostimulatory chemicals on their surface and
therefore explain the current diversification and host range scenario of this pest.
42
CHAPTER THREE
3. 0: EFFECT OF HOST PLANT’S METABOLITES AND SILICA
ON SURVIVAL AND GROWTH OF B. FUSCA LARVAE
3. 1: Introduction
The evolutionary success of phytophagous insects is dependent on their ability to
utilize either many different food plants (generalists) or specialise towards specific
plant species (specialists) (Bernays and Chapman, 1994). Majority of phytophagous
insects are however specialists (Strong et al., 1984; Thompson, 1994) with a narrow
host range but with strong taxonomic conservatism in host plant use (Erhlich and
Raven, 1964). Narrow host ranges of herbivorous insects commonly depend on plant
chemistry including nutrient composition, primary and secondary metabolites as
well as plant physical characteristics (e.g., silica content, spines, and trichomes).
These biophysical plant attributes usually inform the foraging larvae about the
suitability of a plant for feeding (Bernays and Chapman, 1994; Schoonhoven et al.,
1998) and hence are important in the acceptance or rejection of host plants.
Plant chemistry significantly influences insects’ food choice and subsequent
performance (Bernays and Chapman, 1994). Each plant has a unique phytochemical
profile that is detected by the insect and forms the basis of food plant selection and
discrimination (De Boer and Hanson, 1984). However, most of the allelochemicals
present in some plant species are usually active against insects, and renders the plant
repellent, toxic or chemically unsuitable for use as food plant (Rosenthal and
Berenbaum, 1991). However, larvae of many adapted herbivores will tolerate or use
these specific chemical and physical cues to govern the choice of food plant and
43
determine the suitable feeding host plant (Van Loon, 1996). Recent field studies on
the host range indicate that B. fusca larva is highly selective and discriminatory in its
food choice and utilisation (Le Rü et al., 2006a, b; Ong’amo et al., 2006). As is for
the other lepidopteran species, the effects of food on the biology of B. fusca is of
particular importance in understanding host suitability of infested plant species. It is
also important in elucidating the magnitude of injury to the crops attacked which
may accordingly help in designing better economic control strategies for this pest.
Moreover, during host selection, young B. fusca larvae are capable of selecting their
hosts among assemblages of chemotaxonomically dissimilar plants. It has been
suggested that during this selection process, B. fusca is attracted to its specific host
plant within the plant assemblage by blends of volatile and leaf surface metabolites
(Juma, 2005). However, the final decision to accept and feed on the particular
chosen plant, may involve evaluation of the composition of testants present in or on
the plant tissues using larval taste receptors following the first bite. Like for most
other lepidopteran species, development of effective strategies for managing B.
fusca therefore requires a thorough knowledge of the biological interactions of the
larvae and its hosts. A very important component of such interactions is that of
phytochemical basis of host preference for larval feeding and subsequent host
affiliation which is currently lacking for B. fusca larvae. In this Chapter, the
oligophagic feeding behaviour of B. fusca larvae was investigated using some
selected Poacea plants found in pest natural habitats. Similarly the larval feeding to
crude leaf extracts of the plant species used were evaluated to determine the
phytochemical basis of selective feeding and also determine the larval
phagostimulatory allelochemicals present in the plant leaf extracts.
44
Conversely, Poaceous plants often accumulate silica in varying degrees in their leaf
tissues. Level of plant leaf silification often varies directly with larval host plant
discrimination for a number of lepidopteran species studied (Epstein, 1999). The
level of silica in leaf tissues of plants considered were therefore determined to
correlate the level of leaf silification with larval growth and survival. Effect of silica
on both larval development parameters was confirmed using artificial diet amended
with silica. The study focused on both neonates and third instar larvae since neonate
stage is critical for the successful host establishment for feeding while the third
instar is the critical stage at which boring into the stem and extensive damage to
plants occur (Kaufmann, 1983).
3. 2: Materials and Methods
3. 2. 1: Insects
Laboratory-reared B. fusca adults have diminished host location and recognition
responses compared to their wild conspecifics (Calatayud et al., 2008). However, in
a previous experiment, no such relationship was evident between laboratory-reared
and larval progeny from adults collected from the wild but to which wild feral
individuals had been previously added to rejuvenate the colony. Larvae used in this
study were hence sourced from Animal Rearing and Containment Unit (ARCU) of
the International Centre of Insect Physiology and Ecology (ICIPE, Nairobi, Kenya).
Both first and third instars were used for the experiments. Third instars, were
obtained from neonates reared on artificial diet of Onyango and Ochieng’-Odero
(1994). For feeding on intact plants, one day old neonates and freshly moulted third
instar larvae, initially starved for 24 hours prior to the test were used.
45
3. 2. 2: Plants
Maize, Zea mays, wild sorghum, Sorghum arundinaceum, Napier grass, Pennisetum.
purpureum (Schumach.) Arundo donax and Setaria megaphylla were used in this
study. Panicum maximum Jacq. and Panicum deustum Thunb the two other Poaceae
plant species which are frequently found in the natural habitat of B. fusca were also
included in the experiments. Maize was grown in 4-litre pots (one plant per pot)
from seeds provided by Simlaw, Kenya Seeds Company, Nairobi. All the other plant
species were obtained from their natural vegetation in Kenya and propagated from
tillers or stem cuttings in 4-litre pots (one plant per pot) in a greenhouse at ICIPE.
The environmental conditions for growth were approximately 31/17°C (day/night)
with 12:12 hr (L: D) photoperiod. Plants were watered three times weekly and once
with a complete nutrient solution. Three weeks old plants were infested with
neonates while five weeks old plants were infested with third instar larvae. Because
of slow growth compared to other plant species, S. arundinaceum plants were
infested after the 5th and 7th weeks of growth with neonates or third instars larvae,
respectively.
3. 2. 3: Survival and growth of B. fusca larvae on different plant species
Potted plants were carefully and randomly assigned in a greenhouse avoiding
contact among the neighbouring plants. A thin layer of petroleum jelly was applied
on the borderline of each pot to prevent escape, predation or exchange of larvae
among the plants tested. Each potted plant was infested with either thirty neonates
using a fine camel hair brush or five third instar larvae. Preliminary experiments
indicated that any of the seven plant species used could well support an average
46
number of similar larval population of either the two larval stages used. Both larval
instars were weighed prior to plant infestation.
Evaluation of larval performance (growth and survival) was done after, 7, 19 and 31
days following infestation by neonates and 7 and 19 days for third instar larvae. The
weight gain and the number of surviving larvae recovered per plant were recorded.
The percent larval recoveries per plant (corresponding to the percentage of survival)
were also calculated. The relative growth rates (RGR) were calculated by
subtracting the initial larval weight from the final weight and dividing the difference
by the number of days of infestation (Ojeda-Avila et al., 2003). Infestations of
plants with larvae were replicated eight and twelve times for third instars and
neonates respectively.
3. 2. 4: Preparation and extraction of plant leaf powders
For determination of plant sugars or total polyphenol levels, young leaves of
between 4-7 weeks old potted plants were randomly sampled around 10 a.m. for
homogeneity. Leaves were cut into small discs and immediately freeze-dried. The
weight of leaves prior and after freeze-drying was recorded and the moisture content
of each sample evaluated as the difference between the fresh and the freeze-dried
weights. Dried leaf discs were then ground into a homogeneous powder in a blender
and then stored at -20°C in sealed plastic bags in the dark prior to being used for
chemical analyses or bioassays.
Plant leaf powders were extracted first in hexane and then in methanol to remove
compounds of different polarities. Two grams portion of leaf powder of each plant
species was first extracted for 24 hours in 100 ml pure hexane solvent (Aldrich, 99.8
47
%). The extract solution was then filtered and the residual material re-extracted
using the same solvent and procedure. Both extracts (i.e. filtrate) were pooled and
termed as hexane extract. Excess hexane in the residual material was then air-dried
off. Thereafter, the same residual material was re-extracted for another 24 hours in
100 ml solution of methanol and water (3:1v/v). After filtration, the residual
material was again re-extracted using the same solvent and similar procedure. Both
extracts (i.e. filtrate) were again pooled and termed as methanolic extract. The post
extraction residual material was finally discarded.
Thereafter, for each plant species, the filtered extracts were each concentrated under
reduced pressure in a rotary evaporator (Büchl, Switzerland) at 40°C to obtain a
solid crude extract. Based on the freeze-dried weights and the percentage of
moisture content of the foliage collected, solid crude extracts were re-dissolved in a
similar volume of their respective solvent system to give a final solution of
concentration equal to the one found in each plant leaves at the time of collection in
the field. Prior to the bioassays, the methanolic extract was divided into 2 equal
portions A and B: portion A was used for feeding tests while portion B was used for
the analysis of sugars and polyphenol content. All extracts were stored at -20°C until
required for bioassays.
3. 2. 5: Feeding preference tests
Each plant extract was applied on cylindrical pieces of Styrofoam carrier matrices
(surrogate stems) and tested for feeding activities by means of a laboratory bioassay
based on the modified method of Ma and Kubo (1977). Styrofoam carrier matrices
48
(13 mm diameter, 35 mm length) used, were obtained from a 35 mm thick
Styrofoam board by corking with a 10mm diameter cork-borer (Figure3. 1).
Figure 3.1: An illustration showing the preparation of Styrofoam cylinders (13 mm
diameter, 35 mm long) used as feeding matrix for third instar Busseola fusca larvae
to which plant extracts were applied during larval feeding bioassay.
The bioassay arena consisted of a Petri dish (90 mm diameter) lined with filter paper
moistened with distilled water to maintain internal humidity. For all bioassays, an
aliquot of 400 µl of each of the plant extracts or solvent (as controls where
applicable) were topically applied on the inert Styrofoam carrier matrix. Treated and
control bioassay matrices were placed on aluminium foil, air-dried at room
temperature prior to randomly arranging them in the afore-prepared bioassay arena
(Petri dish). Then, one third instar larva was introduced into each bioassay arena and
a Petri dish cover replaced to prevent evaporation of the extract or escape of the
larva.
49
The phagostimulatory activities of third instar larvae to plant‘s extracts were
examined both in non-choice and multiple-choice situations. However, similar tests
were unsuccessful for neonates because they were unable to bite the Styrofoam
matrices onto which the extracts were applied. Choice tests included binary, four
and 8-choice experiments. Both hexane (non-polar) and methanol (polar) extracts
(i.e. portions A of methanolic extracts) were used for all the bioassay tests except in
8-choice tests in which only methanol extracts were used.
For single choice bioassays (Figure 3.2a), Styrofoam carrier substrates were first
impregnated with either hexane or methanolic extracts and tested singly in the
bioassay arena. Control treatments run simultaneously, consisted of Styrofoam
substrate impregnated with the solvent alone (hexane or methanol) and tested singly
in the bioassay arena. In binary choice bioassays (Figure 3.2b), larval preferences
for carrier substrates treated with either methanol or hexane extracts were tested
together in the same bioassay arena. In 4-choice bioassays (Figure 3.2c), both
hexane and methanolic extracts were tested in presence of their corresponding
solvent controls in the same bioassay arena. In 8-choice tests (Figure 3.2d), carrier
substrates containing each of the seven plant methanolic extracts and a control
treated with methanol solvent were simultaneously presented to a single larva in a
similar bioassay arena. In all choice tests Styrofoam carrier matrix were
differentiated by labelling them with different colour codes prior to submission for
bioassay. Each extract impregnated Styrofoam carrier matrix was weighed prior to
submission for all bioassay experiments.
Each bioassay experiment lasted for 36 hours in the dark at 25°C and approximately
80% r.h. and a L12:D12 photoperiod. After feeding, the weight of each Styrofoam
50
carrier matrix was recorded and the feeding response index (FRI) (Cai et al., 2002)
calculated as follow:
FRI (%) = [(X-Y) /X] x 100
Where, X and Y are the weight of the Styrofoam matrix before and after assay,
respectively. Each bioassay was replicated 30 times except for 8-choice condition,
which was replicated 87 times.
Figure 3.2: Illustrations for bioassay feeding setups for third instar Busseola fusca
larva: single choice (A) dual choice (B) 4-choice (C) and 8-choice feeding tests (D).
A
C D
B
51
3.2.6: Analysis of simple sugars in plant leaf extracts
Since the hexane plant extracts did not stimulate feeding in either non-choice or
choice bioassay tests, only the phagostimulatory methanol extracts of each plant
species were subjected to HPLC analysis to determine the stimulatory components
present in the extracts. Therefore, twelve (12) millilitres of portion B of the
methanolic extracts of each plant species was further concentrated in a rotar vapour
at 40°C to remove excess methanol. The resulting aqueous solution was freeze-dried
and the solid re-dissolved in distilled water to give a solution of approximately 5
mg/ml. A modified method of Gomez et al. (2002) was used to purify crude sugar
extracts prior to analysis. Briefly, 0.2 g of polyvinylpyrollidone (PVP) was added to
12 ml of crude extract and thoroughly vortexed. The mixture was then centrifuged at
15000 g at 4°C for 30 minutes. The supernatant was recovered using 4 ml syringe
and sequentially filtered, first through a C18 cartridge and then through Whatman
PTFE membrane filter (0.45 µm). The resulting solution was again freeze-dried and
stored at -20°C prior to sugar analyses.
Fifty milligrams of each of the purified and dried plant sugar material was re-
dissolved in 1 ml of distilled water and the resulting solution filtered on 0.45 µm
PTFE membrane filters prior to loading an aliquot of 2 µl into the HPLC column.
All analyses were performed on a Shimadzu auto-sampler chromatograph
(Shimadzu Kyoto, Japan) equipped with a quaternary pump and an UV/visible
photodiode array detector. Separations were achieved on a supelcosil-NH2 analytical
column (Supelco) (5 µm x 250 mm x 4.6 mm). Isocratic solution with a mobile
phase of acetonitrile and water (3:1v/v) was performed at a flow rate of 1.2 ml/min
(6.2 mPa) and the eluent monitored at 240 nm at 35°C over 25 minutes. The
52
identification of the sugars was achieved by comparing their retention time and UV
spectrum to those of authentic standards (Sigma–Aldrich Chemie Gmbh, Steinheim,
Germany) and confirmed by co-injection. About 90% of the compounds detected by
chromatographic analysis were identified. The quantification of the sugars was
based on peak areas. All solvents used in the analysis were of HPLC grade (Fisher
Scientific, UK).
3.2.7: Analysis of total phenolic compounds in plant leaf extracts
Ten millilitres aliquot of the portion B of the methanolic crude extract of each plant
species was centrifuged at 5500 g for 20 min at 4°C and the supernatant recovered.
A modification of the Folin-Ciocalteau method (Torres et al., 1987) was used to
determine the total content of phenolic compounds in the methanolic fractions.
Briefly, 100 µl supernatant of each plant extract was mixed with 5.9 ml distilled
water and 1 ml of 1 N Folin reagent added. Within five minutes following the
addition of Folin reagent, 2 ml of 20% sodium bicarbonate solution was added,
mixed and the solution incubated for one hour. Optical densities were measured in a
2 ml cell on a Beckman DU 640 spectrophotometer set at 730 nm. The amount of
total phenols in the samples were calculated from a calibration curve generated
using anhydrous gallic acid standards, and were expressed as the amounts of gallic
acid equivalent.
53
3.2.8: Dose response feeding experiments with sucrose and turanose
The effect of sugars (sucrose and turanose) on the feeding response of third instar
larvae were examined using varying concentrations of pure sugar standards (Sigma-
Aldrich, Steinheim, Germany). In no-choice feeding conditions, sugar (sucrose or
turanose) concentrations used ranged between 0 to 0.1 M, corresponding to the
minimum and maximum concentration range obtained among the plant species
analysed. Sugar solutions for dose response experiments were prepared in a mixture
of methanol and water (3:1 v/v) as the solvent. Moreover, similar concentration (0.1
M) of sucrose is used to prepare the artificial diet used to rear B. fusca larvae
(Onyango and Ochieng’-Odero, 1994). To determine the synergistic effects of
sucrose and turanose on larval feeding, a mixture of solutions of both sugars each in
the following proportions were tested: 0 M sucrose/0.1 M turanose, 0.025 M
sucrose/0.075 M turanose, 0.05 M sucrose/0.05 M sucrose, 0.075 M sucrose/0.025
M turanose and 0.1 M sucrose/ 0 M turanose. As already described, 400 µl of either
sugar solution were topically applied on styrofoam carrier matrix and submitted for
feeding bioassay in both single (15 replicates for dose-curve responses and 24 for
the synergistic effect tests) and 6-choice situations (24 replicates).
3.2.9: Extraction and analysis of silica in leaves of different plant species
For each plant species used, young fully expanded leaves were thoroughly cleaned
with ultra pure water and subsequently harvested. Leaves were sliced into small
pieces, put into plastic containers and freeze dried in stoppering tray drier
(Labconco-Germany). Dried leaves were milled into a fine powder befoe digestion.
54
The dilute hydrofluoric acid (HF) extraction and spectrometric molybdenum-yellow
method of Saito et al. (2005) was used for the analysis of the level of silica in the
leave tissues. Briefly, 100 mg of dry weight of each plant powder was digested in 2
ml of HF solution of concentration 1.5 M HF - 0.6 M HCl in 10 ml plastic bottle.
Digestion was carried out at room temperature for one hour with occasional stirring
(after each 10 min). After digestion, 8 ml of distilled water was added to each
sample tube and the resulting mixture homogenised by vortexing before settling
down for further 20 minutes. Silica powder was obtained by heating 50 ml of pure
sodium silicate (Sigma-Aldrich, 338443) at 950°C for 2 h in a furnace before
cooling and grinding the solid into a fine powder. Thereafter, 1mg/ml silica stock
solution was prepared by digesting 0.1 g of silica powder in 20 ml of a 0.3 M HF -
0.12 M HCl solution and then topping to 100 ml with distilled water. This stock
solution was diluted to prepare standard solutions of concentration ranging between
0 to 1 mg/l.
For spectrophotometric determinations of the amount of silica in the powdered leaf
samples, 100 µl aliquot of each plant digest was transferred to a 10 ml plastic tube
and 2ml of 0.1M boric acid solution added. Two millilitres of the molybdenum
solution (0.025 M Mo - 0.4 M H2SO4 - 0.25 M BH3O3) was then added, mixed and
allowed to stand for five minutes to allow the complete formation of molybdenum
yellow complex. Four millilitres of 0.1 M citric acid was then added and thereafter
the mixture vortexed. Similar procedure was used to prepare the aforementioned
silica standards. Optical densities of both samples and standard solutions were
measured in a 5 ml cell on a Beckman DU 640 spectrophotometer at 400 nm
between 4-10 minutes following the addition of 0.1m citric acid. The amount of
55
silica in the samples was calculated from a calibration curve generated using
standard solutions. All reagents and solutions for this experiment were prepared in
silica free polypropylene plastic ware previously soaked in 0.1% HF solution.
3.2.10: Survival and growth of larvae on silica amended diets
To evaluate B. fusca survival and larval growth rates to increasing dietary silica
levels, the artificial diet prepared by Onyango and Ochieng–Odero (1994) was
amended with varying levels of silica (from the aforementioned silica powder
obtained from sodium silicate). The levels of silica in the artificial diet ranged
between 0 to 80 mg/ml and paralleled the concentration range found among the
plant species analysed in this study. Once prepared, 20mls of each diet fraction (with
different silica levels) was dispensed in each heat-sterilised glass vials (7.5 cm long
x 2.5 cm diameter), 9 vials per each silica level. Preweighed B. fusca neonates were
then inoculated into each vial containing the diet fraction (4 neonates per vial)
twenty four hours following hatching. For all experiments, vials were tightly fitted
with a cotton wool following each inoculation and kept in 80% relative humidity on
a 12:12 hr (L/D) photoperiod. Larvae were allowed to feed ad libitum during the
experimental period and the respective diet fractions replaced as necessary.
Thereafter, surviving larvae were counted and weighed after 19 days of development
(the minimum period at which significant effect of silica on B. fusca performance
was discernable).The percent larval recoveries per vial (corresponding to the
percentage of survival) were calculated. The relative growth rates (RGR) were
calculated by subtracting the initial larval weight from the final weight and dividing
the difference by the number of days of development (Ojeda-Avila et al., 2003).
56
3.2.11: Statistical analysis
Data on percent larval recovery per plant were arcsin transformed. Untransformed
results are presented in Tables. All means were separated by Fisher’s PLSD or
Student-Newman-Keuls test following one-way analysis of variance (ANOVA). For
data on feeding preference bioassays in single-choice conditions, ranks were
generated following the Kruskal-Wallis test, using Proc RANK of SAS 9.1 (SAS
Institute, 2003), and means separated using Tukey’s Studentized range test (Proc
GLM) (SAS Institute, 2003). For multiple choices feeding tests data, means were
separated using Wilcoxon signed ranks test (Stat View software, Abacus Concept,
USA). Data on plant sugar content and their relative proportions in the extracts were
log and arcsin transformed respectively while data on total phenolic content per
plant species were also log transformed. Untransformed data are presented in the
Tables. All means were separated by Fisher’s PLSD test following ANOVA. A
linear-regression of feeding response index in relation to the concentration of each
sugar (sucrose or turanose) per extract was done and tested using ANOVA.
Data on plant silica content and percent larval recovery per plant were log and arcsin
transformed, respectively. Untransformed results are presented in tables. All means
were also separated by Fisher’s PLSD test following ANOVA. Linear-regressions of
silica contents on percentages of larvae recovered and RGR (Relative Growth Rate)
were done to evaluate the respective R2 values and thus the relationships between
the silica content and the percentages of survival and RGR. In addition, a linear-
regression of percentage of survival (arcsin transformed) and RGR in relation to the
concentration of silica in the artificial diet was done and tested using ANOVA. All
statistical tests were done using Stat View software (Abacus Concept, USA).
57
3.4: Results
3.4.1: Effect of host plants on the survival and growth of B. fusca larvae
The survival and growth of B. fusca neonates on intact plants varied significantly
among the plant species tested (Table3.1 ANOVA: F6,77 = 119.1, P < 0.0001; F6,77 =
87.7, P < 0.0001 and F6,77 = 122.9, P < 0.0001 after 7, 19 and 31 days, respectively).
Following infestations, maize and wild sorghum supported the greatest larval
survival and growth: 33 and 44% larvae survived on maize and wild sorghum after 7
and 19 days respectively. However, after 31 days of infestation, whereas 41% of
larvae survived on maize, only 10% survived on wild sorghum. In contrast, the other
five plants; P. purpureum, A. donax, P. maximum, P. deustum and S. megaphylla
were poor larval hosts, and a rapid decline in the larval survival on these plants was
obtained over the infestation period tested. In addition, no larva survived on P.
desteum and S. megaphylla beyond seven days following infestation.
Similarly, growth of neonates was significantly higher on maize and wild sorghum
(Table 3.1, ANOVA: F6,59 = 29.8, P < 0.0001; F4,49 = 34.8, P < 0.0001 and F4,34 =
276.2, P < 0.0001 after 7, 19 and 31 days, respectively). Nevertheless, while larval
growth remained significantly higher on maize throughout the infestation period, a
significant decrease in growth was observed on wild sorghum after 19 and 31 days.
Larval growth was significantly lower on the other five plant species, regardless of
the infestation period. A similar trend in both survival and growth was observed
when all the plants were similarly infested with third instar larvae (Table 3.2). As
expected, highest survival was recorded on maize and wild sorghum (ANOVA: F6,47
= 26.8, P < 0.0001 and F6,47 = 18.9, P < 0.0001 after 7 and 19 days respectively).
Except for A. donax on which the growth rate of third instar larvae was significantly
58
lower after 7 days of infestation, growth rates on other plant species did not vary
significantly over the infestation period (ANOVA: F4,24 = 4.0, P = 0.0119). No
variation in growth rate was obtained after 19 days of infestation on Z. mays, S.
arundinaceum or P. maximum, the only three plant species on which third instar
larvae were recovered (ANOVA: F4,12 = 0.6, P = 0.5743), after the experiment
59
Table 3.1: Percentage of surviving (mean1 ± SE, n=12) and growth rates (mg d-1, mean2 ± SE) of Busseola fusca larvae recovered after
7, 19 and 31 days of infestation by neonates on different Poaceae plant species
Plant species After 7 days After 19 days After 31 days
% larvae
recovered
Growth rate % larvae
recovered
Growth rate % larvae
recovered
Growth rate
Z. mays 38.9 ± 1.2 e 0.15 ± 0.012 c 44.5 ± 3.0 c 1.8 ± 0.22 c 41.1 ± 2.9 c 4.51 ± 0.15 c
S. arundinaceum 33.3 ± 1.0 d 0.13 c 42.8 ± 3.7 c 0.4 ± 0.04 b 10.0 ± 1.4 b 1.45 ± 0.12 b
P. purpureum 10.6 ± 1.6 c 0.09 ± 0.013 b 12.2 ± 1.6 b 0.6 ± 0.12 b 2.8 ± 0.9 a 0.07 ± 0.01 a
A. donax 7.8 ± 2.2 bc 0.05 ± 0.013 a 6.1 ± 0.9 a 0.1 ± 0.06 a 2.2 ± 0.7 a 0.21 ± 0.01 a
P. maximum 1.7 ± 0.5 a 0.06 ± 0.002 a 5.0 ± 1.3 a 0.1 ± 0.02 a 0.6 ± 0.4 a 0.04 a
P. deustum 6.7 ± 1.3 bc 0.03 ± 0.004 a 0 a - 0 a -
S. megaphylla 3.9 ± 1.2 ab 0.03 ± 0.005 a 0 a - 0 a
-
Means within a column followed by different letters are significantly different at 5% level (1Fisher’s PLSD test or 2Student-Newman-
Keuls test following ANOVA).
60
Table 3.2: Percentages of surviving third instar Busseola fusca larvae recovered
(mean ± SE, n=8, except for Z. mays and S. arundinaceum n=7) and growth rates
(mg d-1, mean ± SE) after 7 and 19 days of infestation on different plant species
Plant species After 7 days After 19 days
% larvae
recovered
Growth rate % larvae
recovered
Growth rate
Z. mays 85.7 ± 5.7 c 16.4 ± 1.9 b 65.7 ± 9.5 c 12.8 ± 0.4
S. arundinaceum 57.1 ± 9.2 b 10.4 ± 1.5 b 25.7 ± 5.7 b 11.6 ± 1.2
P. purpureum 20.0 ± 7.6 a 10.5 ± 2.3 b 0 a -
A. donax 12.5 ± 5.3 a 3.5 ± 1.2 a 0 a -
P. maximum 20.0 ± 6.5 a 10.4 ± 2.9 ab 5.0 ± 3.3 a 11.9 ± 1.8
P. deustum 0 a - 0 a -
S. megaphylla 0 a - 0 a -
Means within a column followed by different letters are significantly different at 5%
level. (Student-Newman-Keuls test following ANOVA).
3.4.2: Influence of plant metabolites on the performance of B. fusca larvae
Hexanic and methanolic extracts of the seven Poaceae plant species used in this
study were tested for their phagostimulatory activities on third instar B. fusca larvae.
The results of the feeding tests expressed in terms of feeding response index (FRI)
are shown in the Tables 3.3 to 3.6. In all feeding bioassay tests, all plant methanolic
used generally induced significant phagostimulatory activities as compared to the
hexane extracts. Under no-choice conditions, significant higher RFI values were
obtained for larvae fed on methanolic extracts as compared to hexane extracts
61
regardless of the plant species used (Table 3.3). This result was confirmed when
extracts were further tested under 2- or 4-choice conditions (Tables 3.4 and 3.5).
However, not all the RFI values obtained for methanolic extracts under no-choice
conditions were significantly different when compared. In contrast, under 8-choice
conditions, Z. mays extract was clearly the most bioactive among the methanolic
extracts used as demonstrated by the significantly higher FRI obtained compared to
the other plant extracts tested. Extracts of S. arundinaceum, A. donax and P.
maximum, exhibited intermediate but statistically similar feeding response indices.
Moreover, extracts of P. deustum and S. megaphylla were the least bioactive and
exhibited significantly less phagostimulatory activity among all the methanolic
extracts tested.
62
Table 3.3: Feeding response indices (%, mean ± SE, n=30) for third instar Busseola
fusca larvae after 36 hours of feeding on crude leaf extracts of different plant species
topically applied on Styrofoam carrier matrices under no-choice conditions
Plant species Type of extracts Feeding response index
Control Methanol alone 0.7 ± 0.2a
Hexane alone 1.0 ± 0.4a
Z. mays Methanol extracts 8.2 ± 1.2d
Hexane extracts 2.2 ± 0.8a
S. arundinaceum Methanol extracts 4.6 ± 0.8bcd
Hexane extracts 2.3 ± 0.9abc
P. purpureum Methanol extracts 3.8 ± 1.2abc
Hexane extracts 1.9 ± 0.8a
A. donax Methanol extracts 4.4 ± 0.4cd
Hexane extracts 1.9 ± 0.4ab
P. maximum Methanol extracts 6.2 ± 1.4bcd
Hexane extracts 1.1 ± 0.3a
P. deustum Methanol extracts 6.6 ± 1.3cd
Hexane extracts 1.0 ± 0.2a
S. megaphylla Methanol extracts 5.7 ± 1.2bcd
Hexane extracts 1.9 ± 0.7a
Means within a column followed by different letters are significantly different at 5%
level (Tukey’s Studentized Range test).
63
Table 3.4: Feeding response indices (%, mean ± SE, n=30) for third instar Busseola
fusca larvae after 36 hours of feeding on crude leaf extracts of different plant species
topically applied on Styrofoam carrier matrices under dual choice conditions
Plant species Type of extracts Feeding response index
Z. mays Methanol extracts 6.4 ± 1.0b
Hexane extracts 1.5 ± 0.5a
S. arundinaceum Methanol extracts 7.5 ± 1.0b
Hexane extracts 2.8 ± 0.8a
P. purpureum Methanol extracts 4.3 ± 0.9a
Hexane extracts 2.7 ± 0.9a
A. donax Methanol extracts 9.3 ± 1.1b
Hexane extracts 1.8 ± 0.6a
P. maximum Methanol extracts 7.1 ± 1.3b
Hexane extracts 0.5 ± 0.1a
P. deustum Methanol extracts 8.6 ± 1.1b
Hexane extracts 0.9 ± 0.3a
S. megaphylla Methanol extracts 7.2 ± 1.3b
Hexane extracts 1.2 ± 0.4a
Means within a column and plant species followed by different letters are
significantly different at 5% level (Wilcoxon signed ranks test).
64
Table 3.5: Feeding response indices (%, mean ± SE, n=30) of third instar Busseola
fusca larvae after 36 hours of feeding on crude leaf extracts of different plant species
topically applied on Styrofoam carrier matrices under 4-choice conditions
Plant species Type of extracts Feeding response index Z. mays Methanol alone 0.6 ± 0.2a Hexane alone 0.6 ± 0.1a Methanol extracts 5.9 ± 1.1b Hexane extracts 0.9 ± 0.2a S. arundinaceum Methanol alone 0.6 ± 0.1a Hexane alone 0.9 ± 0.2ab Methanol extracts 8.3 ± 1.3c Hexane extracts 1.6 ± 0.5b P. purpureum Methanol alone 0.6 ± 0.2a Hexane alone 0.8 ± 0.4a Methanol extracts 3.6 ± 0.8b Hexane extracts 0.6 ± 0.4a A. donax Methanol alone 0.3 ± 0.1a Hexane alone 0.8 ± 0.2b Methanol extracts 4.7 ± 0.8c Hexane extracts 0.7 ± 0.2ab P. maximum Methanol alone 1.2 ± 0.3a Hexane alone 1.4 ± 0.4a Methanol extracts 8.6 ± 1.5b Hexane extracts 1.4 ± 0.4a P. deustum Methanol alone 0.4 ± 0.1b Hexane alone 0.5 ± 0.2b Methanol extracts 5.2 ± 0.7c Hexane extracts 0.1 ± 0.05a S. megaphylla Methanol alone 1.4 ± 0.6ab Hexane alone 0.4 ± 0.1a Methanol extracts 2.1 ± 0.8b Hexane extracts 1.2 ± 0.6a Means within a column and plant species followed by different letters are
significantly different at 5% level (Wilcoxon signed ranks test).
65
Table 3.6: Feeding response indices (%, mean ± SE, n=87) of third instar Busseola
fusca larvae after 36 hours of feeding on crude methanol extracts of different plant
species topically applied on styrofoam carrier matrices under 8-choice conditions
Type of extracts Feeding response index
Methanol alone 0.7 ± 0.1a
Z. mays 4.5 ± 0.6d
S. arundinaceum 2.5 ± 0.4c
P. purpureum 1.8 ± 0.3c
A. donax 2.5 ± 0.4c
P. maximum 2.0 ± 0.3c
P. deustum 1.2 ± 0.2b
S. megaphylla 1.2 ± 0.2b
Means within a column followed by different letters are significantly different at 5%
level (Wilcoxon signed ranks test).
Nine naturally occurring sugars were identified in the phagostimulatory fractions of
the methanolic plant extracts following HPLC analysis. These included
monosaccharide (xylose, fructose, glucose and galactose) and the disaccharides
(sucrose, turanose, maltose, lactose and melibiose). Sucrose was the most abundant
sugar in each of the plant extracts analysed and varied from 22 to 65µg/mg
(corresponding to 0.06 to 0.2 M), of dry leaf weight (Table 3.7). This represented a
relative proportion of between 25 to 38% of all the sugar content in all the plant
extracts analysed (Table 3.8). The level of sucrose in the extracts however, did not
vary significantly among the plant species studied. Glucose and fructose (possibly as
66
hydrolysis products of sucrose hydrolysis) were also abundant in the extracts. Their
relative proportions were similarly constant among most of the plant species tested,
except, in S. arundinaceum extracts where their levels were both significantly higher
(Table 3.7). Turanose, a disaccharide of glucose and fructose, was the most and
significantly variable sugar component of the extracts tested with its levels varying
from 5 to 36µg/mg (corresponding to 0.01 to 0.1 M) of dry leaf weight. The relative
proportion of this sugar was however lower in Z. mays, S. arundinaceum and P.
purpureum but relatively higher in A. donax, P. maximum, P. deustum and S.
megaphylla(Tables3.7and3.8).
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Table 3.7: Amount of different sugars (µg/mg of leaf dry weight, mean ± SE, n=5) found in leaves of the plant species studied
Plant species Xylose Fructose Glucose Galactose Sucrose Turanose Maltose Lactose Melibiose
Z. mays 2.1 ± 0.4 8.9 ± 2.6a 15.5 ± 4.9a 1.9 ± 0.9 25.7 ± 11.2 4.6 ± 1.6a 5.3 ± 2.1 3.7 ± 0.9 1.7 ± 0.4
S. arundinaceum 1.7 ± 0.6 27.0 ± 7.3b 41.5 ± 11.3b 15.8 ± 10.3 65.1 ± 17.6 10.3 ± 2.7a 5.6 ± 1.6 1.8 ± 0.8 3.9 ± 1.5
P. purpureum 3.3 ± 1.6 15.3 ± 6.6a 16.6 ± 4.4a 2.0 ± 1.7 32.6 ± 16.5 9.1 ± 1.8a 5.1 ± 0.4 3.0 ± 1.0 2.7 ± 0.6
A. donax 2.6 ± 0.7 13.1 ± 3.1a 15.5 ± 3.2a 2.7 ± 0.6 42.0 ± 8.8 33.9 ± 8.1b 16.6 ± 2.9 3.0 ± 0.7 4.4 ± 1.1
P. maximum 1.1 ± 0.4 9.9 ± 3.2a 17.8 ± 5.5a 3.7 ± 1.8 22.4 ± 6.8 18.0 ± 5.5ab 2.8 ± 0.9 2.3 ± 0.6 3.4 ± 1.5
P. deustum 2.1 ± 0.6 7.3 ± 2.4a 17.1 ± 6.0a 4.9 ± 1.2 32.7 ± 12.5 36.0 ± 12.7b 9.4 ± 7.1 7.5 ± 4.8 3.3 ± 1.1
S. megaphylla 1.1 ± 0.4 7.4 ± 0.5a 14.2 ± 5.1a 4.4 ± 1.3 27.9 ± 7.5 28.2 ± 8.8b 2.6 ± 1.1 2.1 ± 0.9 3.8 ± 1.6
Means within a column followed by different letters are significantly different at 5% level (Fisher’s PLSD test). No letter was assigned
when P>0.05 for ANOVA.
68
Table 3.8: Relative proportion of sugars (%, mean ± SE, n=5) found in the dry leaves of the plant species studied
Plant species Xylose Fructose Glucose Galactose Sucrose Turanose Maltose Lactose Melibiose
Z. mays 4.3 ± 1.5 13.7 ± 1.6b 23.2 ± 2.4c 2.6 ± 0.7 31.3 ± 4.9 7.0 ± 1.3a 7.7 ± 1.4b 6.2 ± 1.1 3.8 ± 1.3
S. arundinaceum 1.2 ± 0.3 15.8 ± 0.7b 24.0 ± 1.4c 6.5 ± 3.0 38.4 ± 2.2 6.8 ± 1.1a 3.4 ± 0.5a 1.5 ± 0.6 2.4 ± 0.7
P. purpureum 5.3 ± 2.9 16.0 ± 2.1b 18.9 ± 2.1bc 1.6 ± 1.1 32.3 ± 6.6 11.5 ± 3.2a 6.5 ± 1.5b 4.4 ± 2.2 3.4 ± 1.1
A. donax 2.1 ± 0.8 9.7 ± 2.1a 11.4 ± 2.0a 2.1 ± 0.5 31.6 ± 6.0 25.0 ± 4.6b 12.5 ± 1.7b 2.4 ± 0.7 3.2 ± 0.6
P. maximum 3.0 ± 1.5 12.5 ± 1.9ab 19.7 ± 2.8bc 5.0 ± 1.5 26.8 ± 3.6 20.3 ± 2.4b 3.9 ± 1.2a 3.8 ± 1.5 4.9 ± 1.9
P. deustum 3.5 ± 1.6 7.6 ± 1.5a 14.3 ± 2.0a 5.1 ± 1.2 25.7 ± 5.3 26.5 ± 3.8b 7.2 ± 3.0b 6.1 ± 1.9 3.8 ± 1.0
S. megaphylla 1.5 ± 0.9 10.6 ± 2.6a 15.3 ± 2.2ab 4.7 ± 0.7 30.1 ± 2.6 29.0 ± 3.0b 2.7 ± 1.2a 2.5 ± 1.3 3.6 ± 1.1
Means within a column followed by different letters are significantly different at 5% level (Fisher’s PLSD test). No letter was assigned
when P>0.05 for ANOVA.
The concentrations of total phenolic compounds estimated in plant leaf extracts
ranged between 1.6 to 5.1 µg/mg of dry leaf weight (Table 3.9) and were
significantly varied among the seven plant species used. Zea mays and P.
purpureum had similar although significantly lowest levels, while P. desteum, P.
maximum and S. arundinaceum had the highest levels. However, the concentration
of total phenolic compounds in the methanol extracts did not correlate with the
feeding response indices of B. fusca larva
Table 3.9: Content of total phenolic compounds (in µg gallic acid equivalents/mg of
dry leaf weight, mean ± SE, n=10) found in leaves of the plant species studied
Plant species Total phenolic content
Z. mays 1.8 ± 0.1 a
S. arundinaceum 4.4 ± 0.1 d
P. purpureum 1.6 ± 0.05 a
A. donax 3.6 ± 0.3 c
P. maximum 4.1 ± 0.2 d
P. deustum 5.1 ± 0.2 e
S. megaphylla 2.7 ± 0.1 b
Means within a column followed by different letters are significantly different at 5%
level (Fisher’s PLSD test).
By increasing sucrose concentration, a significant and positive dose-response curve
was obtained for feeding response index (FRI) under no-choice conditions (F1, 73 =
45.128, P<0.0001; Figure 3.3a). In contrast, when the concentration of turanose was
increased, a significant negative dose-response curve was obtained under no-choice
conditions (F1,73 = 6.115, P = 0.0157; Figure 3.3b). Similarly, when a homogeneous
70
mixture of sucrose and turanose were tested for feeding, significantly higher RFI
values were obtained as the level of sucrose in the mixture was increased
proportionally as compared to turanose levels (Table 3.10). In contrast, FRI
decreased significantly with a concomitant increase in the proportion of turanose in
the mixture. Similar trends were obtained under 6-choice conditions (Table 3.11).
Figure 3.3: Dose-response curves for relative feeding indices (mean ± SE, n=15)
obtained by increasing sucrose (A) or turanose (B) levels under no-choice
conditions.
A
B
71
Table 3.10: Feeding response indices (%, mean ± SE, n=24) for third instar
Busseola fusca larvae after 36 hours of feeding on mixture of sucrose and turanose
of varying concentration topically applied on styrofoam carrier matrices under no-
choice (A) and 6-choice conditions (B).
Feeding response index (FRI)
Sugar mixtures tested A B
Methanol alone 2.2 ± 0.5a 1.0 ± 0.4a
0.1 M sucrose / 0 M turanose 15.4 ± 1.5c 5.5 ± 0.9b
0.075 M sucrose / 0.025 M turanose 10.3 ± 2.4b 4.7 ± 1.0b
0.05 M sucrose / 0.05 M turanose 3.6 ± 1.2a 5.6 ± 1.1b
0.025 M sucrose / 0.075 M turanose 5.2 ± 1.2ab 1.4 ± 0.4a
0 M sucrose / 0.1 M turanose 1.3 ± 0.2a 0.4 ± 0.1a
(A) aMeans within a column followed by different letters are significantly different
at 5% level (Tukey’s Studentized Range test). (B): Means within a column followed
by different letters are significantly different at 5% level (Wilcoxon signed ranks
test).
3. 4. 3: Influence of plant silica on survival and growth of B. fusca larvae
Silica content present in the potted plant leaves varied significantly among the seven
plant species analysed (Table 3.11, F7, 248 = 82.8, P < 0.0001). Zea mays and S.
arundinaceum had the lowest silica levels and varied between 20 to 24 µg/mg of dry
leaf weight (corresponding to a concentration of 20 to 24 mg/ml of silica as
determined in the artificial diet). P. maximum and P. deustum harboured the highest
levels of silica in their leave tissues that varied between 45-55 µg/mg
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(corresponding 45 to 55 mg/ml in the artificial diet). S. megaphylla, P. purpureum
and A. donax showed similar but intermediate levels of silica (Table 3.11).
Table 3.11: Amount of silica (mean ± SE, n=34) found in different Poacea plant
species used in the study
Plant species Silica content (µg/mg of dry weight)
Z. mays 19.8 ± 1.0a
S. arundinaceum 24.2 ± 0.3b
P. purpureum 29.3 ± 1.1c
A. donax 29.6 ± 1.2c
P. maximum 45.4 ± 1.1d
P. deustum 55.0 ± 2.3e
S. megaphylla 28.1 ± 0.6c
Means followed by different letters are significantly different at 5% level (Fisher’s
PLSD test following ANOVA).
Plant silica content negatively correlated with percentages of survival or relative
growth rates (RGR) of B. fusca larvae when intact plants were infested by neonates
(Table 3.1). The highest silica containing plant leaves supported the lowest
percentage of larval survival or growth rates. Linear-regressions using the same data
(Table 3.1) showed significant negative correlations (with % survival after 7 days of
feeding: R=-0.766, F1,82 = 43.3, P < 0.0001; with % survival after 19 days of
feeding: R= -0.602, F1,82 = 46.5, P < 0.0001; with % survival after 31 days of
feeding: R= -0.455, F1,82 = 21.4, P < 0.0001; with RGR after 7 days of feeding: R= -
0.596, F1,62 = 34.1, P < 0.0001; with RGR after 19 days of feeding: R= -0.335, F1,54 =
6.8, P = 0.0119 and with RGR after 31 days of feeding: R=-0.477, F1,37 = 10.9, P =
73
0.0021). For example, P. deustum with the highest silica content was the least
preferred plant for larval survival and growth while Z. mays with the least silica
content was mostly preferred plant for larval survival and growth. Similarly, plant
silica content negatively correlated with percentages of survival when all the plants
were infested by third instar larvae (Table 3.2), with highest silica containing leaves
supporting the lowest percentage of larval survival. Linear regression results using
the same data (Table 3.2) showed significant negative correlations with percentage
of survival (with % survival after 7 days of feeding: R= -0.432, F1,52 = 12.0, P =
0.0011; with % survival after 19 days of feeding: R= -0.337, F1,52 = 6.7, P = 0.0126).
Nevertheless, no correlation was evident between RGR and the levels of leaf
silification when the plants were infested by third instar larvae (Table 3.2).
A significant variation in % survival and growth rates were obtained when larvae
were allowed to feed on artificial diets amended with increasing levels of silica
(results of ANOVA: F4, 40 = 5.1, P = 0.0020 for % survival; F4, 40 = 31.9, P < 0.0001
for RGR). A strong interdependence was also obtained between silica content and B.
fusca larval survival and growth rates after 19 days of development (Figure 3.4). A
significant negative correlation existed between the level of silica and the
percentages survival and the relative growth rates over the feeding periods tested (R
= -0.482, F1, 43 = 13.0, P = 0.0008 with % survival; R = -0.855, F1, 43 = 117.6, P <
0.0001 with RGR). Highest % survival and growth rates were recorded among
larvae fed on low-silica containing diets (0 to 20 mg/ml), while lowest % survival
and growth rates were observed among larvae fed on high-silica containing diets (60
to 80 mg/ml).
74
Figure 3.4: Dose-response curves for percentage of surviving larvae recovered (arcsin
transformed values, mean ± SE, n=9) (A) and relative growth rates (RGR) (mg.d-1, mean ±
SE, n=9) (B) obtained after increasing silica concentration.
A
B
75
3. 5: Discussion
For phytophagous insects, plants can be grouped into three distinct classes: (i) hosts
fed upon in nature; (ii) acceptable non-hosts, not fed upon in nature but which can
act as food plants in the laboratory; and (iii) unacceptable hosts, neither fed upon in
nature nor in the laboratory (Gupta and Thorsteinson, 1960; De Boer and Hanson,
1984). All the seven plant species used in this study influenced significantly the
survival and growth rates of both first and third instar B. fusca larvae in laboratory
conditions. Both larval developmental stages performed better on Z. mays and S.
arundinaceum throughout the infestation period as compared to the rest of other
plant species used. The suitability of Sorghum sp. and Z. mays on the performance
of B. fusca has been well reported previously (van Rensburg et al., 1989; Haile and
Hofsvang, 2002; Le Rü et al., 2006a). However, although neonate survival and
growth on the other plant species tested especially P. purpureum, A. donax and P.
maximum was inferior to that of Z.mays and S.arundinaceum, marginal survival
recorded on the three plant species suggest the ability of larvae to include not only S.
arundinaceum but also other related wild plants as food source, probably when the
preferred hosts are not available. Marginal utilisations of wild plants by B. fusca
larvae have also been observed in the field particularly in seasons when maize or
cultivated sorghum are absent (Ong’amo et al., 2006). Growth rates of Lepidoptera
on their host plants are influenced by many factors, but primarily nutritional
composition and secondary plant compounds (Herms and Mattson, 1992; Slansky,
1992). Therefore, minimal larval performance on wild plants used in this study may
suggest the presence of antibiotic properties possibly due to the presence of plant
76
secondary metabolites, or similarly these plants lacked nutrients necessary for
optimal growth or were physically less suitable for feeding by the larvae.
The acceptance of a particular host plant for feeding by phytophagous insect’s larvae
is dependent on the phytochemical blends of the plants, which may induce
phagostimulant or deterrent bioactivities. Compounds present in both methanol and
hexane extracts variably affected feeding responses of third instar larvae. B. fusca
larvae were stimulated to feed more on methanolic extracts than on hexane extracts
indicating that most chemical feeding stimulants were more polar and soluble in
methanol. This result is consistent with similar studies in which polar compounds
extractable in methanol have been shown to elicit positive feeding responses in
Helicoverpa species (Lepidoptera: Noctuidae) (Cai et al., 2002). In the contrary,
hexane extracts elicited aversive or non-remarkable larval feeding responses
suggesting that non-polar compounds (e.g. lipids) present in hexane extracts were
non-phagostimulatory (as exhibited in no-choice experiments) and less attractive (as
exhibited in multiple-choice experiments) to the foraging larvae. Aversive feeding
responses therefore suggest either the hexane extracts contained chemicals that
deterred feeding or were toxic to the larvae. However, the fact that no larval
mortality was observed after feeding on hexane extracts indicated the presence of
deterrent rather than toxic allelochemicals in the hexane plant extracts.
The relative contribution of each of the plant’s methanolic extract on larval feeding
response and preference was however evident in the data obtained for 8-choice
bioassay tests. Larvae responsiveness to compounds in the crude extracts varied
among the plant species used. Significant feeding response was observed for Z.
mays extract than for any other plant species used. The hierarchical ranking order of
77
larval feeding preference on methanolic plant extracts from the most preferred to the
least preferred was: Z. mays > S. arundinaceum = P. purpureum = A. donax = P.
maximum > P. deustum = S. megaphylla. This rank ordering paralleled survival and
growth rates on intact plants, where larvae fed on Z. mays and S. arundinaceum,
exhibited the best performance as compared to the residual survival and growth rates
on P. purpureum, A. donax and P. maximum. Similar hierarchical ordering of plants
according to preference has been observed in the field for B. fusca and the species
has been reported to use a host lower in the hierarchy especially when the preferred
species is not available (LeRü et al., 2006b). The flexibility in host choice
presumably enhances the ability of individuals of B. fusca to cope up with variation
in the environment as has been suggested by Thompson and Pellmyr (1991).
The discriminatory behaviour of B. fusca larvae towards some plants used in this
study appeared to be influenced by the quantitative and qualitative chemical
characteristics of each particular plant species used. The general feeding preferences
on methanolic extracts as compared to hexane extracts suggested the presence of
soluble phagostimulatory compounds in the methanol extracts. Subsequent isolation
and identification of the chemical constituents in the methanolic extracts of each of
the plant species used revealed the presence of nine main soluble sugars including
xylose, fructose, glucose, galactose, sucrose, turanose, maltose, lactose and
melibiose. Most of these sugars identified have been implicated to stimulate feeding
in many lepidopteran larvae (Lopez and Lingren, 1994; Bartlet et al., 1994). Sucrose
represented the major sugar component in all plant methanolic extracts analysed
regardless of the plant species. A significant positive dose-response curve for
feeding response index as a function of increasing concentration of sucrose,
78
confirmed the phagostimulatory effect of sucrose towards B. fusca larvae. Similar
phagostimulatory activity of sucrose to larval feeding has been reported for a
number of other lepidopteran species (Albert et al., 1982; Bartlet et al., 1994;
Bowdan, 1995).
Apart from sucrose, turanose was the most variable sugar component in most of the
plant extracts analysed. High levels of this sugar were present in plant methanol
extracts of P. deustem, A. donax and S. megaphylla. Extracts from the three plant
species mediated minimal larval survival and growth rates and therefore levels of
this sugar correlated negatively with B. fusca larval feeding responses. A similar and
significant negative dose-response curve of RFI expressed as a function of
increasing levels of pure turanose standards, corroborated the phagodeterrent effect
of turanose towards feeding by larvae. This was contrary to a similar dose response
curve of RFI as a function of increasing sucrose concentration that exhibited
positive correlations with larval feeding. Turanose which is structurally analogous to
sucrose is a reducing disaccharide with α-(1, 3) glycoside bond between glucose and
fructose. It is therefore possible that the opposing effect of turanose and sucrose to
larval feeding lies in the small but subtle structural differences inherent in the two
sugars that are likely to give rise to variation in molecular fit in the larval taste
receptors inducing the opposing behavioural responses. Though the role of turanose
in feeding of lepidopteran larvae is limited in the literature, the same sugar has been
demonstrated to possess a weak phagostimulatory potential for corn earworms,
Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) (Lopez and Lingren, 1994).
Turanose has similarly been demonstrated to act as a strong phagostimulant for the
red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae) (Vander
79
Meer et al., 1995). Moreover, both sugars appeared to have a synergistic effect on B.
fusca larval feeding responses when presented together in a solution mixture applied
on Styrofoam carrier matrices especially when turanose was weakly concentrated.
This implies that the host discriminatory ability and the selective feeding behaviour
of B. fusca larvae may partly depend on the relative concentration of each of the two
sugars in the plant tissue and this could sufficiently explain the oligophagous
feeding habits of B. fusca larvae.
Plant total phenolic compounds have been reported to correlate negatively with
performance of most important phytophagous species (Haukioja et al., 1985;
Bergvinson et al., 1994; Santiago et al., 2005). Despite, their possible defensive
roles against herbivores, some soluble phenolic compounds have been reported to
possess strong phagostimulatory properties to lepidopteran larvae (Torto et al.,
1991). The amount of total phenolic compounds in leaf extracts studied varied
significantly among the plant species tested. P. deustum, P. maximum and S.
arundinaceum extracts had the highest concentrations of total phenolic compounds
as compared to the other four Poaceae plant species analysed. However, in contrast
to sucrose or turanose, the levels of total phenolic compounds in the methanolic
extracts did no correlate with the feeding response indices or the growth and
survival of B. fusca larvae. For example, while both S. arundinaceum and P.
maximum had similar and high levels of total phenolic compounds, larvae fed more
on the methanolic extracts of the former than of the later. It is therefore possible that
the determination of total phenolic compounds (i.e. pooled compounds) does not
allow correlation of the importance of a particular phenolic compound present in the
entire plants with the insect survival and performance parameters.
80
Silica in plants can constitute between 0.1 to 10% weight of the dry matter of most
plant species (Ma and Takahashi, 2002). Results obtained from this study, indicate
that the levels of amorphous silica varied considerably among the plant species
analyzed. The silica content was about three times greater between the highest (P.
deustum, 55.0 ± 2.3 µg/mg) and the least (Z. mays, 19.8 ± 1.0 µg/mg of dry leaf
weight) following the analysis of silica from samples digested in dilute HF
Nevertheless, levels of silica present in all plant leaf tissues sampled appeared to be
within the concentration ranges as has been previously reported in similar studies
(Ma and Takahashi, 2002).
Differences in silica content among plants are frequently associated with plant
resistance to insect attacks (Keeping and Meyer, 2002; Kvedaras et al., 2005;
Keeping and Meyer, 2006). Therefore, during host selection process herbivorous
insects may discriminate between high- and low-silica plants, and feed preferentially
on the later. Growth rate of B. fusca larvae on the tested plant species correlated
negatively with the degree of plant leaf silification. Larvae preferentially fed on Z.
mays and S. arundinaceum foliage as exhibited by increased in growth rates and
high larval survival percentages on both plants over the whole infestation period.
Similar performance indices were however poor on other wild grasses especially P.
deustum and P. maximum, that had the highest level of endogenous silica in their
leaf tissues. This clearly indicated that plants analyzed in this study had different
capacities to accumulate amorphous silica in their leaf tissues and high levels of
silica in the leaves correlated negatively with the feeding and developmental
performance of B. fusca larvae. This was further confirmed by using artificial diets
amended with increasing levels of silica. It was evident that the detrimental effect of
81
silica on B. fusca larval performance is dose dependant and as little as 20 mg/ml of
silica in the artificial diet was sufficient enough to disturb feeding and development
of B. fusca larvae. For example, larval RGR decreased significantly from 1.06 ±
0.05 to 0.86 ± 0.06 mg d-1 following incorporation of 4grammes of silica into
200mls of artificial diet. This further corroborated the negative role of silica on the
feeding and development of B. fusca larvae and parallels results of similar studies
that previously sought to correlate the effect of silica on the feeding behaviour of
several lepidopteran stemborer species (Peterson et al., 1988; Kvedaras et al., 2005;
Keeping and Meyer, 2006). For B. fusca, such correlation can form the basis of host
plant/artificial diet discrimination and may indicate that silica content in plants is a
reliable predictor of food choice by B. fusca larvae.
The deterrent effect of silica that resulted in the reduction in the numbers and
growth rates of larva on high silica containing plants may partly be attributed to the
effect of silica at both nutritional and physical levels. As reported by Panda and
Kush (1995) elevated levels of silica in plant cell wall might increase the bulk
density of the diet and discourage larvae from ingesting sufficient quantities of
nutrients and water leading to their poor performance. While compensatory feeding
behaviour on nutritionally poor diets have been reported for some insects, feeding of
B. fusca larvae on similar artificial diets amended with silica did not alter the rate of
diet consumption as was reflected by the concentration dependent diminutive larval
mass gain over the entire feeding period. Similar results have been reported by
Keeping and Meyer (2006), Kvedaras and Keeping (2007) and Kvedaras et al.
(2007b). Moreover, other studies have indicated that most specialist herbivores have
limited ability to increase their diet consumption to compensate for feeding on poor
82
quality plants (Lee et al., 2003). B. fusca larvae can therefore be considered more
specialist than generalist (Le Rü et al., 2006a, b; Ong’amo et al., 2006) and this
could have given rise to the greater impact of silica on larval performance.
Despite of the deduced evidence on the importance of silica in deterring host plant
choice by B. fusca larvae, the underlying mechanism of silica mediated resistance is
still speculative and may only support the hypothesis that silica impedes larval
penetration of host plants. It was observed that most larvae fed on the relatively
high silica amended diets could not easily bore into the diet material but only fed
superficially, nor bore into the stems of highly siliconized plants. This provides
initial insight into the possible mechanisms of silica mediated resistance against B.
fusca larvae and provides a correlative support for a physical role of plant silica in
impeding feeding of larvae. However, this observation does not preclude the
probable induction of biochemical defence pathways facilitated by the presence of
soluble silica following wounding of tissues by B. fusca larvae.
From an applied point of view the fact that silica augments the resistance of various
plants to pest insects (Keeping and Kvedaras, 2008) and that plants have a capacity
to accumulate silica (Ma and Yamuji, 2006), is particularly relevant for silica
deficient soils of cereal growing regions of sub-Saharan Africa. Silica amendments
in plant tissues may provide improved resistance to B. fusca in these regions. This
has been demonstrated for Sesamia calamistis (Hampson) (Lepidoptera: Noctuidae)
following Z. mays tissue silification (Sétamou et al., 1993) as a result of silica
addition to the soils. However, for B. fusca, field trials are required to confirm this
hypothesis and also confirm the quality of the modified crops for human
consumption following silification of their tissues.
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CHAPTER FOUR
4. 0: PHYSIOLOGICAL ADAPTATIONS AND GENETIC
DIVERSITY OF BUSSEOLA FUSCA LARVAE
IN RELATION TO HOST PLANT USE
4. 1: Introduction
The diversity of phytophagous insects is thought to be the result of the evolutionary
process of host specialisation (Jaenike, 1990; Bernays and Chapman, 1994). Studies
on host range and utilisation by B. fusca appears to be restricted and specific (Le Rü
et al., 2006a). The principal host plants for this species are cultivated cereal crops
especially sorghum and maize (Le Rü et al., 2006a, 2006b; Ong’amo et al., 2006).
Although, the species is well adapted to the two important staple crops, it is also
reported to marginally attack other Poaceous non-crop plant species, including
Sorghum arundinaceum, Pennisetum purpureum, Arundo donax and Cymbopogon
nardus. Sorghum arundinaceum, A. donax and Cymbopogon nardus appear the most
preferred non-crop host plants for B. fusca among the currently known candidate
wild plant species. For example, of the 346 B. fusca larvae recovered on wild plants
in Ethiopia, 328 (95%) were found on A. donax (Le Rü et al., 2006a), suggesting a
strong association of B. fusca with the particular plant species.
To exploit a number of food plants but with diverse allelochemicals predictably
encountered for growth and which are not in normal repertoire of larva’s diet, young
lepidopterous larva often depend on their digestive performance especially in
enzyme assortment in the gut system (Brattsten, 1991; Peric-Mataruga et al., 1997).
For example, food composition has been reported to modify and alter the activity of
84
insect digestive enzymes (Hinks and Erlandson, 1994; Lazarevic and Peric-
Mataruga, 2003). Therefore, the complement of digestive enzymes induced after
larval foraging on chemotaxonomically diverse plants should reflect the
allelochemical composition of the particular plant species. Such plastic responses to
nutritive stresses are important for predicting insect’s outbreaks and understanding
mechanisms of host plant specialisation.
It was hypothesized by Sezonlin et al. (2006) that B. fusca abandoned its ancestral
Poaceous wild hosts, switched and colonised the current and possibly nutritionally
superior cultivated hosts, following their domestication (S. bicolor) or introduction
(Z. mays) into Africa. Like many other phytophagous insects, adaptation of B. fusca
to these new cultivated host plants following switch from their native wild hosts
may have been accompanied by larva’s physiological and/or behavioural
adjustments due to the process of natural selection (Futuyma and Moreno 1988).
Since host choice and oviposition behaviour among phytophagous insects are
genetically determined (Jaenike, 1990), natural selection is likely to have favoured
oviposition by B. fusca to hosts that currently support better growth and survival of
the offspring as suggested by Gassmann et al. (2006).
Phenological, phytochemical and morphological differences among host plants may
also promote utilization of specific hosts following host shifts and may also explain
the current trend of host choice and affiliation by B. fusca. Sustained and prolonged
colonization of adapted plants may induce the selection of adaptive traits and
genetic differentiation within insect populations utilising specific hosts (Rice, 1987;
Diehl and Bush, 1989; Jaenike, 1990) and may subsequently evolve into host races
(Bush, 1994; Prowell et al., 2004). In phytophagous insects, species with narrow
85
host range are more prone to genetic differentiation than insect species with a wide
host range (Futuyma and Moreno, 1988, Peterson and Denno, 1998). Host
associated genetic differentiation has been documented in a number of moth families
including Noctuidae (Pashley, 1986; Leniaud et al., 2006; Ong’amo et al., 2008),
Tortricidae (Emelianov et al., 1995), Prodoxidae (Groman and Pellmyr, 2000) and
Crambidae (Martel et al., 2003; Leniaud et al., 2006).
Although geographical differentiation of B. fusca populations have been reported
(Sezonlin et al., 2006), little is known about the possible genetic isolation of the
species by host plant. As Fox (1993) and Forister (2005) pointed out, differences
between populations or species of insects in preference for and performance on a
plant are usually genetically controlled, there could be a possible existence of
genetic differentiation between B. fusca larva feeding on cultivated crops and those
found to feed on wild plants such as A. donax.
The aim of this study was therefore to determine the larval physiological response in
terms of digestive enzyme assortment induced in larvae fed on leaves of different
plants and also to determine the possible existence of genetic differentiation between
two B. fusca populations collected from cultivated crops (Z. mays) and A. donax in
Kenya and Ethiopia respectively using cytochrome b gene markers. The
mitochondrial cytochrome b gene was studied because it is informative at the
intrageneric level in Lepidoptera (Simmons and Weller, 2001). It was envisaged that
the two candidate plant-associated strains represent populations which differ
genetically as a result of host specialisation.
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4. 2: Materials and Methods
4. 2. 1: Insects
Larvae used in the study were sourced from laboratory reared individuals from
Animal Rearing and Containment Unit (ARCU) of the International Centre of Insect
Physiology and Ecology (ICIPE, Nairobi, Kenya). Neonates for experiments were
directly used after one day following hatching. For molecular studies, B. fusca
larvae were collected directly from Z. mays and A. donax plants in Kenya and
Ethiopia respectively. Sampling was simultaneously done in both countries because
neither country could provide both host plants infested with B. fusca larvae at the
time of sampling. The Kenyan population were sampled from maize plants in
Machakos and Mt. Kenya regions while the Ethiopian larvae were sampled from A.
donax plants in Abay Mado and Baimraar regions. Five sampling points (localities)
were considered for each region sampled. Plants with symptoms of stem borer
infestation were dissected in situ for larval recovery. Plants from which larvae were
recovered were first identified and the recovered larvae kept separately with respect
to host plant species and site of collection. Larvae were then reared on artificial diet
of Onyango and Ochieng’- Odero (1994) to adult moths. However most larvae
sampled from A. donax did not develop to adulthood (only two fully developed to
adulthood) and were therefore only used after attaining the fifth larval stage and
prior to slipping into diapauses. Larvae and adult moths obtained after rearing were
thereafter placed individually into 30 ml plastic vials and preserved in absolute
ethanol (> 99%) at -20°C prior to DNA extraction.
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4. 2. 2: Plants
Potted Maize, Zea mays, wild sorghum, Sorghum arundinaceum, Napier grass,
Pennisetum purpureum (Schumach.) Arundo donax, Setaria megaphylla, Panicum
maximum and Panicum deustum were used in this study. These plants were infested
with neonate larva at the age of between 3-7 weeks of growth.
4. 2. 3: Determination of activities of digestive enzymes in larval homogenates
of larvae fed on leaves of different plant species.
Dietary exposure to plant allelochemicals can induce physiological responses in
neonate larvae that may consequently determine the feeding behaviour of late
instars. A fast semi-quantitative analysis of enzymatic activities present in the larvae
previously fed on leaves of different plant species was performed using the API-
ZYM system (BioMérieux, Marcy l’Etoile, FRA) as described by Rahbé et al.
(1995). Approximately 100 neonates were used to infest each of the seven plant
species used. Surviving larvae were recovered from each of the infested plants 4
days following infestation. Preliminary experiments indicated that four days of
growth was the optimum period in which a good number of surviving larvae could
be recovered from all the seven plants species tested. Due to the small size of the
larvae, it was not possible to isolate the midgut from the rest of the body tissues for
gut enzyme analysis. Therefore, a total of forty larvae with decapitated heads (to
avoid contamination with the salivary enzymes) collected from each plant species
were separately homogenized in 1,300 µl distilled water and centrifuged (18,000 g,
10 min., + 4°C). A volume of 65 µl of the resultant supernatant was pipetted to each
of the twenty porous plastic micro-cup of the API system (i.e., 19 micro-cups
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dispersed with the substrate and one without the substrate (as the control) and the
plate incubated (37°C for 4 h). A similar procedure was used for neonates samples
previously fed only on water over the 4 days growth period which were used as the
control. Enzyme activities were detected by cleavage of a chromogenic substrate
(naphtyl derivatives) dispersed dry on a porous plastic micro-cup (∼100 µl).
Enzymatic reactions were enhanced by an SDS-based acid Tris buffer (Zym A)
applied as a drop in each well after incubation of the plate. The reactions were
visualized after addition of Fast Blue BB solution (Zym B) to each well. The level
of activity was determined by visually assigning a rank value (0-5) corresponding to
the intensity of colour produced by each well after comparison with the colour scale
provided with the kit (0-5, from ≤ 5 to ≥ 40 nmoles of substrate released) (Figure
3.1). Rank values reflecting enzyme activity from naïve water-fed neonate larvae
served as baseline; such that the rank values of this group were subtracted from the
rank values obtained for the experimental groups to yield the feeding score value.
Score values greater than zero indicated induction of appreciable enzyme activities
in the larval homogenate.
The following nineteen enzyme activities commonly found in the guts of
lepidopteran larvae were analysed for their presence using the API-ZYM kit system
as described above : alkaline phosphatase (pH 8.5), esterase (C4), esterase lipase
(C8), lipase (C14), leucine aminopeptidase, valine aminopeptidase, cystine
aminopeptidase, trypsin (N-benzoyl-DL-arginine-2-naphtylamidase), α-
chymotrypsin (N-glutaryl-phenylalanine-2-naphtylamidase), acid phosphatase (pH
5.4), naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-
89
glucuronidase, α-glucosidase, α-glucosidase, N-acetyl-beta-glucosaminidase, α-
mannosidase and α-fucosidase.
Ranks for enzyme activities were generated following the Kruskal-Wallis test, using
Proc RANK of SAS 9.1 (SAS Institute, 2003), and their means separated using
Tukey-Kramer test (Proc GLM) (SAS Institute, 2003). Linear regression analysis
was performed to determine the relationships between enzyme activities and neonate
performance (percent survival or larval growth rates) on the plant species studied
over the infestation period.
4. 2. 4: DNA isolation and purification
For DNA extraction, a total of 23 individual larvae (14 from Z. mays and 9 from A.
donax) were randomly selected from 41 B. fusca specimens sampled. Total genomic
DNA was extracted from each of the individual thoracic muscles using commercial
kit (DNeasyTM Tissue Kit, Qiagen Cat # 69506 GmbH, Germany) protocol with
Proteinase K digestion as recommended for animal tissues. Briefly, each moth was
taken out of the ethanol dried well and the segment containing the thorax removed,
cut into smaller pieces and put into 1.5 ml microfuge tubes. Samples were then
crushed under ice using electric mortar and pestle. To the crushed samples, 180 µl of
lysis buffer (ATL) and 20 µL of proteinase K were added and mixed thoroughly by
vortexing. The DNA samples were then incubated for three hours at 55°C in a
shaking water bath, after which, they were removed and vortexed for another 15s.
To this sample, 200 µL of AL buffer was added and the solution thoroughly, mixed
by vortexing prior to incubating for another 10 min at 70°C. A similar amount of
absolute ethanol (98 %) was added following incubation and the samples were again
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thoroughly mixed prior to pipetting into a DNeasy Mini Spin column placed in a 2
ml collection tube before spinning for a minute at 6000 x g. The precipitate was
replaced into another DNeasy spin column, washed with 500 µl wash buffer1
(AW1) by spinning for a minute at 6000x g. The DNA precipitate was then eluted
by pipetting 100 µl of elution buffer directly onto the DNeasy membrane in the spin
column, previously washed with 500 µl wash buffer 2 (AW2) for 3 minutes at 20000
x g. The DNA samples were collected in clean 1.5 ml microfuge tube and then
incubated at room temperature for 1 minute and then span for another 1 minute at
6000 x g. The purified DNA was stored at -20°C until required for amplification.
4. 2. 5: Cytochrome b gene amplification and digestion
Polymerase chain reaction (PCR) was used to amplify the 1000 bp Cyt b
mitochondrial fragment using two primers: CP1 (5′-GATGATGAAATTTTGGATC-3′)
(modified from Harry et al., 1998) and Tser (5′-TATTTCTTTATTATGTTTTCAAAAC-3 ′)
(Simon et al., 1994). The PCR was performed on a Biometra GeneAmp PCR
System in a 25 µl reaction mixture containing 1 µl of the genomic DNA, 5X Green
GoTaq® Flexi Buffer, 0.24 mM dNTPs, 3 mM MgCl2, 0.4 µM of each primer and 1
unit of Taq polymerase (GoTaq, Promega). After initial denaturation at 94oC for 5
min, PCR condition was 40 cycles at 94oC for 1 min of denaturation, 46oC for 1 min
30 s of annealing, 72oC for 1 min 30 s of extension and a final extension period of
10 min at 72oC. Amplified products were then digested by an enzyme restriction as
described by Calatayud et al. (2008) as follows. The 25µl reaction mixture
containing 100µg/ml bovine serum albumin (BSA), 0.5U Promega XhoII restriction
enzyme and the amplified DNA incubated for 4 h at 37oC. The digested fragments
91
were visualised by means of electrophoresis on 1.5% agarose gel previously stained
with ethidium bromide. Amplified DNA products were further purified with the
Promega Wizard SV Gel purification kit following the manufacturer’s protocol prior
to sequencing reaction.
4. 2. 6: Cytochrome b gene sequencing and analysis
DNA sequencing reactions and the analysis for the determination of the extent of
genetic differentiation between the two populations were performed at the
laboratoire Evolution, Génomes et Spéciation- CNRS (France). Reactions were
performed using the ABI PRISM® BigDye™ Terminator v3.0 Ready Reaction
Cycle Sequencing Kit (Applied Biosystems), cleaned using ethanol/EDTA
precipitation. Sequences were visualized on an ABI 3130 automated sequencer
using Big-Dye fluorescent terminators. The consensus sequences obtained were
aligned manually using Mac Clade 4.05 (Maddison and Maddison, 2001).
Basic sequence statistics and number of haplotypes were determined using DnaSP
(Rozas et al., 2003). The following parameters were used to estimate genetic
variability among populations between the two host plants (Z. mays and A. donax):
number of haplotypes (h), number of polymorphic sites (S), haplotype diversity (d)
(Nei, 1987), nucleotide diversity (π) (Lynch and Crease, 1990) using the Jukes and
Cantor correction (Jukes and Cantor, 1969). The extent of genetic differentiation
between the populations (FST) (Hudson et al., 1992) was performed with the
Arlequin 2.000 software (Schneider et al., 2000).
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4. 3: Results
4. 3. 1: Activities of digestive enzymes in larval homogenates
Results of the semi-quantitative assay of activities of larval digestive enzymes
induced following neonates consumption of plant foliage over 4 days period of
infestation are given in Table 4.1. Most of the aminopeptidase activities relative to
the activities in the control homogenates (homogenates of naive unfed larvae) did
not increase significantly regardless of the plant leaves ingested by the larvae.
However, activities of most of the sugar degrading enzymes, including β-
galactosidase, β-glucuronidase, α-glucosidase and β-glucosidase increased
appreciably. Highest activities of sugar degrading enzymes were exhibited by larvae
exposed on leaves of Z. mays, S. arundinaceum and P. purpureum. However, the
activity of β-glucosidase was significantly higher in homogenates of larvae fed only
on Z. mays and S. arundinaceum. Conversely, C4 esterase was significantly higher
in neonate larva that consumed leaves of P. maximum, P. deustum and S.
megaphylla. Highest positive correlations between enzyme activities and percentage
survival or relative growth rates of neonates were obtained with α-glucosidases, β-
glucosidases and β-galactosidase over the infestation period. In contrast, negative
correlations between same larval performance parameters and enzyme activities
were obtained for esterase (C4) and esterase lipase (C8) (Figure 4.1).
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Figure 4.1: A representative plate showing differential staining of the API ZYM
plate with ZYM A and ZYM B reagents after 4 hours of incubation of homogenates
of larvae fed on plant leaves with chromogenic substrates dispersed on the porous
plate. Each coloured well was assigned a rank score (0-5) based on the intensity of
colour formed. Differences in rank scores (colour intensities) between the plant leaf-
fed and naïve water-fed neonate larva were used to determine the feeding score
values for each enzyme activity analysed and used for statistical analysis. Negative
control (1), alkaline phosphatase (2), C4 esterase (3), C8 esterase (4), C14-lipase (5),
aminopepetidases (6-11) and glycosidases (12-20).
94
Table 4.1: Semi-quantitative analysis of enzyme activities in the homogenate of entire neonate larvae (with decapitated heads) reared for 4 days on
different plants species. Enzyme activities (means ± SE, n=5) correspond to the activities found in the crushed larval homogenate (40 larvae per
replicate). Activities correspond to the release of 5, 10 and 20 nmoles of substrate per 4 hours of incubation at 37°C (rate 0: no change in activity)
Enzymes Z. mays S. arundinaceum
P. purpureum A. donax P. maximum P. deustum S. megaphylla
Alkaline phosphatase 0 0 0 0 0 0 0 Esterase (C4) 0 0 0 0 0.2 ± 0.2a 1.0a 0.2 ± 0.2a Esterase lipase (C8) 0 0 0 0 0.2 ± 0.2 0 0 Lipase (C14) 0 0 0 0 0 0 0 Leucine aminopeptidase 0 0 0 0 0 0 0 Valine aminopeptidase 0.7 ± 0.2a 0.6 ± 0.2a 1.0a 0.4 ± 0.2a 0 0 0.6 ± 0.2a Cystine aminopeptidase 0 0.2 ± 0.2a 0 0 0.2 ± 0.2a 0.2 ± 0.2a 0 Trypsin 0 0 0 0 0.5 ± 0.3 0 0 α-chymotrypsin 0 0 0.7 ± 0.2a 0 0.5 ± 0.3a 0 0 Acid phosphatase 0 0 0 0 0 0 0 Naphthol-AS-BI-phosphohydrolase
1.0b 0.6 ± 0.2ab 1.0b 1.0b 0.2 ± 0.2a 1.0b 0.8 ± 0.2ab
α -galactosidase 0 1.2 ± 0.5a 0 0 0 0.5 ± 0.3a 0 β-galactosidase 1.2 ± 0.3b 1.2 ± 0.2b 1.7 ± 0.2b 0.8 ± 0.2a 0.5 ± 0.3a 0 0.2 ± 0.2a β -glucuronidase 1.2 ± 0.2a 2.0 ± 0.5b 2.0b 1.0a 1.0a 0 0.8 ± 0.2a α -glucosidase 1.8 ± 0.2b 1.6 ± 0.2b 2.0b 1.2 ± 0.2a 0 0.5 ± 0.3a 1.0 ± 0.3a β -glucosidase 1.8 ± 0.5c 1.4 ± 0.2c 1.0b 0.4 ± 0.2a 0.7 ± 0.2ab 0 0 N-acetyl-beta-glucosaminidase
0 0 0 0 0 0 0
α -mannosidase 0 1.0 ± 0.3a 1.5 ± 0.3a 0 0.7 ± 0.2a 0 0 α -fucosidase 0 0.4 ± 0.2 0 0 0 0 0 Means within a line followed by different letters are significantly different at 5% level (Tukey-Kramer test).
95
Table 4.2: Correlations between enzyme activities and percentages of surviving larva of Busseola fusca recovered or relative growth rates
(RGR) after 7, 19 or 31 days, following infestation by neonates. R-values are given
Dependent variables . Independent variables % survival
after 7 days % survival
after 19 days % survival after
31 days RGR after 7
days RGR after 19
days RGR after 31
days Alkaline phosphatase - - - - - - Esterase (C4) -0.3 -0.4 -0.3 -0.4 -0.2 - Esterase lipase (C8) -0.1 -0.1 -0.1 -0.1 -0.1 - Lipase (C14) - - - - - - Leucine aminopeptidase - - - - - - Valine aminopeptidase +0.2 +0.3 +0.2 +0.3 +0.03 +0.09 Cystine aminopeptidase -0.05 -0.2 -0.1 -0.1 -0.1 +0.05 Trypsin -0.2 -0.1 -0.1 -0.1 -0.2 -0.2 α-chymotrypsin -0.3 -0.2 -0.2 +0.2 - -0.4 Acid phosphatase - - - - - - Naphthol-AS-BI-phosphohydrolase
+0.06 - +0.2 - +0.3 +0.2
α-galactosidase +0.2 +0.1 -0.06 +0.2 -0.08 - β-galactosidase +0.4 +0.5 +0.3 +0.6 +0.2 - β-glucuronidase +0.3 +0.3 +0.1 +0.5 +0.03 -0.09 α-glucoidase +0.5 +0.6 +0.4 +0.5 +0.4 +0.3 β-glucosidase +0.7 +0.6 +0.6 +0.6 +0.3 +0.5 N-acetyl-beta-glucosaminidase
- - - - - -
α-mannosidase - +0.1 -0.2 +0.3 -0.1 -0.3 α-fucosidase +0.2 +0.2 +0.03 +0.3 - +0.07
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4. 3. 2: Population distribution
For the purpose of the analysis of the genetic structure of B. fusca, Z. mays and A.
donax were considered as host plants without quantifying their relative contribution
to population dynamics of the pest in the field. Cytochrome b gene analysis assigned
individuals found on both plant species in two distinguishable clades, that is, clades
KI and KII (FST = 0.45, P < 0.001). Clade KI was characterised by three DNA
fragments (approximately 600, 300 and 100bp) while KII was characterised by two
DNA fragments (880 and 100bp) (Figure 4.2). Individuals from KII clade were more
abundant as compared to the KI clade (Figure 4.2).
Figure 4.2: A representative agarose gel electrophoresis plate of DNA products of
cytochrome b DNA fragment of several Busseola fusca larvae randomly sampled
from Z. mays and A. donax noted from 1 to 9 following PCR amplification and Xho
restriction digestion. Distinct bands were identified using 100bp ladder and results
showed the presence of two mitochondrial clades labelled KI and KII.
900 bp
600 bp
300 bp
200 bp
100 bp
100 bpDNA ladder
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The spatial distribution of the two clades varied among regions sampled. The
Kenyan populations collected from the two localities, Mt. Kenya and Machakos,
belonged exclusively to clade KII (Figure. 4.2), while individuals from the Ethiopian
population belonged exclusively to clade KI (Figure 4.2). Similarly, individuals in
the KII clade were collected from maize while those in clade KI were collected from
A. donax. Results of these clades are summarized in Table 4.3 below. Though, both
clades occurred on different hosts they also occurred in different geographical
regions.
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Table 4.3: Summary of the distribution of Busseola fusca clades with respect to host
plant species and locality
Extraction code Country Locality Host plant Sex Clade
2965 Kenya Mt Kenya Zea mays Female KII
2966 Kenya Mt Kenya Zea mays Female KII
2967 Kenya Mt Kenya Zea mays Female KII
2968 Kenya Mt Kenya Zea mays Male KII
2969 Kenya Mt Kenya Zea mays Male KII
2970 Kenya Machakos Zea mays Male KII
2971 Kenya Machakos Zea mays Male KII
2972 Kenya Machakos Zea mays Male KII
2973 Kenya Machakos Zea mays Female KII
2974 Kenya Machakos Zea mays Female KII
2975 Kenya Machakos Zea mays Female KII
2976 Kenya Machakos Zea mays Male KII
2977 Kenya Machakos Zea mays Male KII
2978 Kenya Machakos Zea mays Male KII
2979 Ethiopia Abay Mado Arundo donax Female KI
2981 Ethiopia Abay Mado Arundo donax Male KI
2982 Ethiopia Abay Mado Arundo donax Larva KI
2983 Ethiopia Abay Mado Arundo donax Larva KI
2984 Ethiopia Abay Mado Arundo donax Larva KI
2985 Ethiopia Abay Mado Arundo donax Larva KI
2986 Ethiopia Abay Mado Arundo donax Larva KI
2987 Ethiopia Baimraar Arundo donax Larva KI
2988 Ethiopia Abay Mado Arundo donax Larva KI
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Table 4.4: Genetic diversity (mean ± SD) of the cytochrome b gene of Busseola
fusca sampled from Z. mays and A. donax from Kenya and Ethiopia
Genetic diversity parameters Kenya (Z. mays) Ethiopia (A. donax)
No. sequences (n) 14 9
No. segregating sites (S) 18 16
Number of haplotypes (h) 4 6
Haplotype diversity (d) 0.5118 ± 0.0370 0.6302 ± 0.0426
Nucleotide diversity (%) 0.3783 ± 0.1956 0.2221 ± 0.1343
4. 4: Discussion
According to Lindroth (1989), food plant chemicals strongly affect the activity of
insect digestive enzymes. The assessment of enzyme activities in the larval
homogenates of larva fed on plant leaf tissue indicated that plant foliage consumed
by the larvae significantly influences the activity of some of the larval digestive
enzymes. Esterase (C4) was significantly induced in larvae fed on P. maximum, P.
deustum and S. megaphylla. In addition, larvae fed on the three plant species had the
poorest growth rates and minimal survival. Induction of esterase has been positively
correlated with increased resistance to allelochemical toxicity in plants in several
Lepidoptera species (Ahmad et al., 1986; Lindroth et al., 1993; Hwang and
Lindroth, 1997). For example, increased esterase activities have been implicated in
the metabolism of toxic phenolic glycosides in several lepidopteran species
(Lindroth and Hemming, 1990; Lindroth and Bloomer, 1991; Lindroth and
Weisbrod, 1991). For instance, the consumption of phenolic glycosides by neonates
of gypsy moth, induced esterase activity and similarly their survival rates were
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negatively correlated with increased enzyme activity (Lindroth and Weisbrod,
1991). Although the high activity of esterase induced in B. fusca larvae fed on P.
maximum, P. deustum and S. megaphylla in this study cannot be directly correlated
with the level of total phenolic compounds in plant’s leaf tissue; it could still be a
possible indicator of the presence of high levels of a specific phenolic glycoside in
the respective plant leaves. It is therefore possible that pronounced activity of
esterase particularly, C4 esterase in neonate larva fed on the three plant species was
a direct physiological response to toxic allelochemicals present in leaves of the three
plant species. In fact, Panicum sp. and Setaria sp. are known to harbour toxic beta-
phenylethylamine alkaloids (such as N-methyltyramine) and a phenolic acid, setarin
(4-allyloxycoumarin), respectively (Steglich et al., 2000). It is therefore likely that
toxic allelochemicals were present in the less preferred plants and could have been
the possible elicitors of the physiological responses in digestive enzymes following
their consumption by neonate larva. Similarly, although Z. mays and S.
arundinaceum were the preferred host plants for larvae in this and other related
studies (Hofsvang et al., 2001), and were therefore likely to contain
phagostimulatory components in their tissues, both plants are reported to also
contain toxic compounds such as DIMBOA or gramine (Niemeyer, 1988) and a
cyanogenic glycoside (dhurrin) (Conn, 1980), respectively. These compounds have
been documented to inhibit the activity of esterase in a number of insect species.
DIMBOA has also been documented to inhibit the activity of serine proteases and
exopeptidases (tryspin, chymotrypsin and leucine aminopeptidases in S.
nanogrioides (Ortego et al., 1996). In the current study, the level of esterase and the
three protease activities were not elevated in the homogenates of larvae that
101
consumed leaves of both plant species. This clearly indicated the possible presence
of these noxious compounds in the two plant species. However, since B. fusca larvae
has for a prolonged time fed on both plant species (coevolved with) it is probable
that larvae of this species developed other efficient enzymatic detoxification
mechanisms or genetically based adaptations to cope up with the noxious
allelochemicals encountered in the two plant species. Similar adaptations to toxic
plant allelochemicals has been reported for example, in the European corn borer,
Ostrinia nubulalis (Hübner) (Lepidoptera: Pyralidae) (Campos et al., 1989) and for
specialised Heliconius caterpillars (Lepidoptera: Nymphalidae) which convert
harmful cyanogenic glycosides into soluble and harmless thiols (Engler et al., 2000).
This biochemical transformation process prevents the later to release cyanide and
allows the caterpillars to use the toxic compounds as a nitrogen source. Therefore, it
is possible that B. fusca larvae have evolved similar mechanisms to cope up with the
noxious chemicals present in both plant species as a result of long and close
association with the two plants.
For a number of lepidopteran species, the presence of sugars in insect’s diets usually
induce the production of sugar-cleaving enzymes such as α�-glucosidases and β-
glucosidases in their guts. The activities of most of the sugar metabolising enzymes
(glucosidases) were much more pronounced in larvae fed on plant leaves that
supported highest larval growth rates and survival. Glucosidases are digestive
enzymes that have a critical role in the final stages of carbohydrate digestion; they
hydrolyse the -o-glycosyl bonds of sugars. In addition, these enzymes have an
important role in plant-herbivore coevolution as they are involved in the catabolism
of plant secondary metabolites (Hemming and Lindroth, 1999; Hemming and
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Lindroth, 2000). In the present study, levels of glucosidases studied were variably
elevated in larval homogenates of larvae fed on leaves of different plant species
considered. This indicated the induction of the particular enzyme activities as a
response to and in the proportion at which the respective sugar substrates were
present in the plant leaf tissues consumed. Indeed identical sugars were found at
various concentrations in the leaf extracts of the same plants analysed in this study.
Among the glucosidases, β-glucosidase was particularly more pronounced among
larvae fed on Z. mays and S. arundinaceum. The activity of β-glucosidase enzyme
has been reported in many organisms (Yu, 1989) and specifically functions to
catalyze biochemical pathways that involve the cleavage of β-linked o-glycosyl
linkages of many glycosides (Clausen et al., 1990). In insects, β-glucosidases play
an important role in the digestion of β-(1, 4) linked sugars (Mendiola-Olaye et. al.,
2000; Zibaee et al., 2008; 2009). Therefore elevated activities of this enzyme in
homogenates of larvae fed especially on Z. mays and S. arundinaceum leaves could
be related to larva’s physiological response to the presence of high levels of β-(1,4)
linked sugars (methyl-β- glycosides and cellobiose) in the plant leaf diets. Since β-
glucosidase is also active on several other glycosides (Zibaee, et al., 2009), its
presence could be important in the biotransformation of DIMBOA in Z. mays among
other harmful glycosides (including cynogenic glycosides, phenols) in sorghum or
other plant species to their harmless aglycones via detoxification reactions. This may
explain the excellent performance exhibited by larvae on both plant species despite
their highest probability of possessing the two harmful allelochemicals.
Moreover, larva fed on Z. mays, S. arundinaceum and P. purpureum had also
appreciable activities of α-glucosidase and β-galactosidase. Alpha-glucosidase,
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catalyses the hydrolysis of maltose, sucrose and trehalose, β-galactosidase hydrolyse
lactose while β-fructofuranosides hydrolyse sucrose and raffinose (Zibaee et al.,
2008; 2009). But unlike larvae fed on S. arundinaceum and P. purpureum, Z. mays
fed larvae did not exhibit any increased level of β-glucuronidase activity. Alpha-
glucosidases are found in the alimentary canal of almost all insect species and
hydrolyze the di- and oligosaccharides. Therefore, elevated activity of this enzyme
may be correlated to the presence of phagostimulatory constituents such as sucrose
or maltose in the plant leaf tissue. Sucrose has been demonstrated to be a key
phagostimulant for many insect species even for B. fusca (Onyango and Ochieng’-
Odero, 1994). Therefore, the positive correlations between α-glucosidase activities
and percentages of surviving larvae recovered or relative growth rates observed
throughout the infestation period confirmed that phagostimulation is a key process
for B. fusca larval survival on a number of plant species.
The activities of larval proteases (aminopeptidases and trypsin) were however lower
than the activities of sugar metabolising enzymes in homogenates of larva fed on
most of the plant species tested. This indicated that B. fusca larva generally and
prefferentially relies on simple sugars other than proteins as their food source. This
may be possible since the evolutionary advantage for host plant specialisation by
herbivorous insects has been hypothesised to involve an increase in energetic
efficiency (Zibaee et al., 2008). Therefore, maize and wild sorghum should have a
more suitable composition, level or ratio of different carbohydrates required by B.
fusca larvae for their optimum performance unlike the other plant species considered
in this study. Similarly, it is likely that S. arundinaceum and other poaceous plants
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may serve as alternative hosts for B. fusca larvae in the absence of Z. mays
depending on the relative proportion of sugars present in the plants’ leaf tissues.
Large fluctuations in host- plant resources can influence insect populations to the
extent that genetic differentiation develops among local populations. However, this
often requires substantial genetic drift and strong (long-term) isolation among
populations (Berenbaum, 1996; Kawecki, 1997). This has been demonstrated in
herbivorous insects that feed on a wide range of host plants: colonization of a new
host may induce the selection of adaptive characters and population genetic
differentiation (Rice, 1987; Diehl and Bush, 1989). Data on genetic diversity of the
DNA samples analysed used in this study indicated a split of B. fusca populations
into two genetically differentiated groups i.e. clade KI and clade KII . Clade KI
populations were found exclusively on wild A. donax while clade KII populations
were found on Z. mays. Individuals found on A. donax (Ethiopian population) were
more variable as compared to the Z. mays (Kenyan) associated population. This was
evidenced by the high number of haplotypes (6) that originated from 9 sequences in
A. donax associated population in contrast to the lower number (4) haplotypes that
originated from 14 sequences of the Z. mays associated population (Table 4.4).
These data was further corroborated by values of genetic diversity parameters (h, d,
π,) obtained following sequence analysis (Table 4.4). The three genetic parameters
indicated that Z. mays sampled population was indeed more stable but genetically
less variable than A. donax sampled individuals.
However, according to this data, it is not conclusive whether the observed
population differentiation can be attributed to correspond to specific host plant use
by larvae since similar genetic demarcation but based on geographical isolation has
105
already been reported (Senzolin et. al., 2006). According to these authors, genetic
sub-structuring of B. fusca populations sampled from maize and sorghum across
three major biogeographic zones in Africa can be demarcated into three
mitochondrial clades; one from Western Africa, and two designated Kenya I and II
(KI and KII), from Eastern and Central Africa. These population units display
ecological preferences along altitudinal gradients. While the West African
population unit preferentially occupy the low altitudes of dry savannah zones (Harris
and Nwanze, 1992), the East and central Africa populations predominantly inhabit
the highlands characterised by wet and cold seasons (Kfir et al., 2002).
A recent and similar extensive sampling of B. fusca larvae on both cultivated
maize/sorghum fields and wild plants from the same Ethiopian regions considered in
this study revealed the coexistence of both population units. Both units do not
display the expected biases in plant host distribution. Additionally, both clades have
been reported to coexist on maize or cultivated sorghum plants in some regions in
the Kenyan Rift Valley. Furthermore, the Kenyan clade (KII) predominantly found
on maize has also been sampled on wild A. donax plants in South Africa. Therefore,
it is possible that genetic differentiation exhibited by B. fusca larvae from larval
samples obtained in both countries corresponds to geographical differentiation other
than host use. It is hence possible that wild hosts and other crops which are
marginally infested by B. fusca could only and possibly serve as part of a larger
refuge to this species especially in seasons when the preferred hosts (maize and
cultivated sorghum) are absent as suggested by Gould, (1998). In the present case,
A. donax a perennial plant could probably function as sink habitat for B. fusca
larvae. The plant could only be marginally infested simply for oviposition by
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females mainly attracted to from the nearby but dwindling preferred hosts especially
during off-seasons, than act as ‘true’ host on which autonomous and large
population can develop over several generations. This is well corroborated by the
inability of A. donax to support survival and growth following its infeststion by
larvae in the laboratory as was evidenced in this study. However, this does not rule
out firsthand the possibility of the existence of host differentiated species among B.
fusca populations.
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CHAPTER FIVE
5. 0: GENERAL DISCUSSION, CONCLUSION AND
RECOMENDATIONS
5. 1: General discussion
Phytophagous insects that damage crops are often polyphagous, feeding on several
types of crops and weeds. Recent field studies indicate that B. fusca exhibit more
oligophagic (Le Rü et al., 2006a, 2006b; Ong’amo et al., 2006) than polyphagic
feeding habits contrary to as previously reported (Polaszek and Khan, 1998; Haile
and Hofsvang, 2001). The host plants for the pest are primarily maize and sorghum
(Haile and Hofsvang, 2001), although a significant number of populations have also
been found on wild Poaceae plants (mostly S. arundinaceum and A. donax) (Le Rü
et al., 2006a, 2006b; Ong’amo et al., 2006). The purpose of this study was therefore
to determine the extent of oligophagy of B. fusca by first examining the sensory
abilities of the larvae to recognise different plants as hosts and its subsequent
performance in terms of survival and growth when fed on the same host plants.
Since larval performance on a particular plant of most lepidopteran species is
depended on the plant’s chemical and physical cues, the study was designed to
determine plant stimuli that influence larval host choice and subsequent
colonisation. The potential existence of host plant associated populations of the
species as was postulated by Senzolin, (2006) was also examined.
The distribution of chemo- and mechanoreceptors on the antennae and maxillae as
sensory equipments modulating host choice and acceptance of B. fusca larvae were
ascertained based on observations made using scanning electron microscopy,
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selective staining with silver nitrate, and gustatory electrophysiological recordings.
The sensilla present on the larval maxillary galeae are typical of other Lepidoptera
family and consist of two uniporous styloconic sensilla that are contact
chemoreceptors, three basiconic sensilla, and two aporous sensilla chaetica
(Faucheux, 1999). The larval maxillary palp is two-segmented and posses eight
small basiconic sensilla at the tip that were found to be gustatory as have been
described for other lepidopteran species (Albert, 2003). The antennae of B. fusca
larvae are short and simple. Similar to other lepidopteran species, the antennal
sensilla comprise of two aporous sensilla chaetica, three multiporous cone-shaped
basiconic sensilla, three small basiconic sensilla and one aporous styloconic
sensillum (Dithier, 1937; Faucheux, 1999). The basiconic sensillum present on the
third antennal segment displayed a contact chemoreception response. However,
similar sensilla on the first and second antennal segments did not show any action
potential activity during tip recording tests and are therefore unlikely to be
associated with chemoreception. Nevertheless, the significant and positive dose-
response electrophysiological curve obtained with an increasing sucrose
concentration for the antennal basiconic sensillum that displayed a contact
chemoreception response confirmed for the first time the presence of gustatory
chemoreceptors on the antennae of a Lepidoptera larva.
The oligophagic feeding behaviour of B. fusca larvae as evaluated based on the
performance of larva on the seven plant species used, indicated that their growth
rates and survival was better on Z. mays and S. arundinaceum. Marginal
performance was also recorded on P. purpureum, A. donax and P. maximum
confirming the oligophagy of the species. Preferential feeding habits of B. fusca
109
larvae on different Poacea plants as exhibited in this study have also been observed
in the field (Le Rü et al., 2006a, 2006b; Ong’amo et al., 2006), further confirming
the oligophagous feeding habits of B. fusca larva.
Therefore, it appears likely that non-crop plants especially wild sorghum, S.
arundinaceum, may also serve as alternative hosts for B. fusca larvae (Polaszek and
Khan, et al. 1998; Haile and Hofsvang, 2001), possibly in the seasons when Z. mays
is unavailable in the field. Marginal survival and growth rates of larvae that was
recorded on P. purpureum, A. donax and P. maximum, some of the hypothesised
ancestral hosts also suggests that B. fusca larva has a residual ability to utilise not
only S. arundinaceum but also other wild plants present in their microhabitat as food
sources in the absence of preferred hosts. Utilisation of several plant species albeit at
different degrees, is suggestive that several plant cues from the tested species
influence the growth and survival of B. fusca larvae.
Plant physical features (trichomes, levels of tissue silification) can easily influence
the choice of host plant use by B. fusca larvae. Levels of endogenous silica among
plants are frequently associated with plant resistance to insect attacks, whereby in
most cases resistance is positively correlated with plant silica content (Meyer and
Keeping, 2005; Laing et al., 2006). Phytophagous insects will therefore discriminate
between high- and low-silica containing plants and preferentially feed on the later
during host selection process. In this study, feeding and survival of B. fusca larvae
on the plant species considered correlated negatively with their silica content.
Larvae preferentially fed on Z. mays and S. arundinaceum foliage with low silica
content as compared to the five other plants used and shown to contain high levels
of silica. Similar results have been reported for a number of lepidopteran larvae
110
(Schoonhoven et al., 2005; Kvedaras et al., 2007a). The resistance effect of silica on
B. fusca larval growth rate and survival was dose dependent as was confirmed by
gradual increase of the amount of silica in silica-amended diets. For B. fusca, such
correlation may form the basis of host plant/artificial diet discrimination and
indicates that silica content in plants is a reliable predictor and contributory factor of
food choice and discrimination for this species.
Host plant discriminations and preferences by B. fusca larvae appear to be partially
determined by plant nutritional factors and/or plant primary/secondary metabolites.
Polar methanol extractable host plant chemical stimulated feeding of third instar B.
fusca larvae, compared to deterrent hexane soluble plant compounds. The relative
suitability of host plant utilisation by B. fusca larva was reflected in the order of
preference of host plants which included Z. mays > S. arundinaceum = P.
purpureum = A. donax = P. maximum > P. deustum = S. megaphylla. This ranking
order reflected the effects of allelochemicals present in particular plant leaves and
their overall contribution larval choice of the host plant. Similarly, feeding responses
of larvae to the host plants’ extracts and fractions thereof also paralleled feeding
responses on intact plants from which the extracts were obtained implying that that
chemical composition are important in controlling food choice behaviour of B. fusca
larvae. More importantly, the abundance and relative proportion of sucrose and
turanose in the host plant leaves appear to determine the basis of plant acceptance or
rejection by larvae. Typical to other Lepidoptera larvae (Sharma, 1994; Bowdan,
1995; Yazawa, 1997), sucrose was the most stimulatory sugar to B. fusca larval
feeding among the sugars identified in the plant extracts. However, turanose, which
was highly variable in its content among the plant leaf extracts, appeared
111
phagodeterrent to larval feeding especially at high concentrations as was confirmed
by the negative dose response curves. The overall effects of the two sugars on
feeding by larvae was reflected in the relative proportion of the specific sugar in the
host plants: plants with high sucrose content stimulated feeding while those with
high turanose content deterred larval feeding. Both sugars however, appeared
synergistic on the feeding responses by B. fusca larvae when presented together in
specific concentrations of each sugar, particularly in the ratio of 3:1 sucrose-
turanose concentration. Therefore the host discriminatory ability and the selective
feeding behaviour of larvae seem to be dependent on the relative concentration of
each of the two sugars in the plant tissue and this may sufficiently explain the
oligophagous feeding habits of B. fusca larvae.
Although the amount of total phenolic compounds in the leaf extracts studied varied
significantly among the plant species tested, their concentrations did not correlate
with the feeding and survival of B. fusca larvae on entire plants. This is contrary to
most studies that positively correlated the level of phenolic compounds with insect
resistance (Haukioja et al., 1985; Bergvinson et al., 1994; Santiago et al., 2005). The
absence of a relationship between the two does not however, rule out first hand the
importance of phenolic compounds in host choice and selection by B. fusca larvae.
This is because the determination of total phenolic compounds (i.e. pooled
compounds) as was carried out in this study, does not allow correlating the
importance of a specific phenolic compound present in the entire plants with insect
performance parameters.
The assessment of the activities of larval enzymes following feeding on different
plants considered in this study using the API-ZYM system (a fast semi-quantitative
112
analysis of enzymatic activities) indicated that the type of plant foliage consumed by
larvae significantly influenced the activity of some of B. fusca digestive enzymes.
C4 esterase was significantly induced in the guts of larvae fed on P. maximum, P.
deustum and S. megaphylla. Moreover, larvae fed on leaves of the three plant
species had equally the poorest growth rates and hardly supported larval survival
beyond 7days of following plant infestations. Therefore, induction of esterase can be
positively correlated with a direct larval physiological response to the toxic
allelochemicals present in the specific plant leaves.
Sugar degrading enzyme activities particularly of β-glucosidases were however
more pronounced in homogenates of larvae fed on Z. mays and S. arundinaceum
plants that supported highest larval growth rates and survival. Moreover, larva fed
on Z. mays, S. arundinaceum and P. purpureum had also a significant increase in the
activities of α-glucosidase and β-galactosidase activities. Elevated activities of these
enzymes may be attributed to the direct physiological response to the presence of
phagostimulatory enzyme substrates present in the respective plant species by the
feeding larvae. The positive correlations among α-glucosidase activities and
percentages of surviving larvae recovered or relative growth rates registered
following 7, 19 or 31 days of infestation by neonates confirmed the importance of
phagostimulation for survival and growth of B. fusca larvae on host plants. The
differential and pronounced levels of glucosidases found in larval homogenates
following larval feeding on a variety of plant leaves may be attributed to the balance
between sucrose and turanose among the other sugars present in the plant leaf
tissues. The activities of proteases (aminopeptidases) were however, lower than the
activities of sugar metabolising enzymes in larval homogenates of larvae fed on all
113
of the plant species studied, indicating that B. fusca larvae generally relies on
carbohydrates than proteins as food source.
Low dispersal ability, geographical barriers, habitat fragmentation as well as host
plant use and availability in the field are considered as key factors responsible for
the genetic differentiation in phytophagous insects (Emelianov, et al. 1995; Mopper,
1996). Data on genetic diversity obtained using cytochrome b genetic marker
indicated the split B. fusca populations into two genetically differentiated groups.
However, genetic substructuring in this species could be attributed to geographical
isolation as reported by Senzolin, (2006) rather than influenced by host use.
5. 2: Conclusion and recommendations
The contributions of the olfactory and gustatory organs in food plant discrimination
were examined in larvae of B. fusca. Larvae of this species have extensive olfactory
and gustatory sensilla that allow for the detection of host plant physical and
chemical stimuli important in host plant recognition and selection. Both sensilla
types are unevenly and unequally distributed on the antennae and the maxillary palp
but appear to be important in host plant choice and selection. The gustatory
receptors which are mainly located on maxillary palp but sparsely on the antennae
are involved in the detection of plant surface chemicals by the help of apical
agryrophilic pores. Nevertheless, mapping of B. fusca larval sensory structures
establishes a basis for the additional electrophysiological and behavioural
investigations that may be important for the fully understanding of the relative roles
of each of the sensory organ (the antennae, maxillary palpi and the epipharyngeal
sensilla) in food plant choice and discrimination for feeding by neonate larvae. Like
114
has been demonstrated for adult moths (Birkett et al., 2006), further studies are
required to determine the ability of young larvae to perceive host plant volatile cues
as well as the type and distribution of sensilla involved in such perceptions.
Additionally, the nature and function of minute sensilla present on the tip of the
maxillary palp of B. fusca larvae remains to be fully elucidated and understood.
The patterns of host plant use and the oligophagic feeding behaviour of B. fusca
larvae as has been reported in a number of field studies (Le Rü et al., 2006a, 2006b;
Ong’amo et al., 2006; Haile and Hofsvang, 2001) and was corroborated in the
current study. Larva of this species are selective in their feeding habits and exhibit a
strong feeding preference for Z.-mays and S.arundinaceum plants, indicating a
restricted host plant range. These larva are also able to use other hosts present in
their ecological niche albeit minimally, possibly in seasons when the preferred hosts
are unavailable. Selective feeding habits of B. fusca larvae appear to be partly
influenced by host plant physical and chemical cues especially plant silica content
and the presence and relative proportion of specific plant sugars.
Similar to other lepidopteran stemborers (Setamou et al. 1993; Schoonhoven et al.,
2005; Kvedaras et al., 2007a), endogenous plant silica influences B. fusca larval
performance. High plant silica content negatively correlated with B. fusca larval
survival and growth. While the identification of the specific mechanisms of silica
resistance to B. fusca larval discriminatory feeding behaviour may not be crucial for
the development of resistant cultivars; such information can facilitate plant breeding
or selection programs. Therefore, future studies focused on the mechanism of silica
resistance could be useful in understanding host plant resistance to B. fusca larvae.
Additionally, comparative studies are needed to establish the extent at which various
115
poaceous plants are able to accumulate silica and subsequently contribute to
resistance to feeding by B. fusca larvae. Such experiments can be carried out by
studying insect performance parameters on plants grown in silica amended soils at
various doses. However, from an applied point of view the fact that silica augments
the resistance of various plants to pest insects (Keeping and Kvedaras, 2008) and
that plants have a capacity to accumulate silica (Ma and Yamuji, 2006), this is
particularly relevant for the silica deficient soils in the cereal growing regions of
sub-Saharan Africa. Therefore, silica amendments for susceptible cultivars in these
regions may provide improved resistance to the insect although field trials are
required to confirm the effects and physiological responses of the plants as regard to
this amendment. Additionally, the effects of plant tissue silifications on human
consumption should be clearly tried and ascertained.
Plant selection and subsequent colonisation by B. fusca larvae is similarly influenced
by the type and levels of some primary and secondary plant metabolites in the plant
leaf tissues. The levels and type of specific metabolites in plant tissues as were
established in this study can vary according to plant species and have been shown to
mediate food choices in a number of insect species (Mohammed et al., 1992; del
Campo and Renwick, 2000; del Campo and Miles, 2003). In the present study
sucrose and turanose were the key sugars isolated and appeared to determine B.
fusca larval food choice. At high concentrations, whereas sucrose was
phagostimulatory, turanose was phagodetterent to larval feeding. Moreover the two
sugars seem synergistic and can positively modulate larval feeding at specific ratio.
The dose response feeding experiments further revealed that B. fusca larvae can
distinguish by taste the presence of both sugars in their diets and hence both or
116
either of the sugars may play specific roles in larval food choice especially from
plant sources. However, future investigations of the relative proportion of each of
the two sugars present in different hosts may reveal a rationale for the selective
management of B. fusca in agricultural fields. Moreover further laboratory based
studies are required to determine the inhibitory mechanisms of turanose to larval
feeding. Including turanose in electrophysiological tip recording tests to establish
turanose sensitive cells and performing a dose response experiments with turanose
amended artificial diets would provide a useful way of determining the role of
turanose in host plant recognition and performance by B. fusca larva. Although both
sugars undoubtedly and greatly contribute to the overall food choice by B. fusca
larva, the importance of some but yet to be identified metabolites that mediate
phagostimulatory/deterrent larval responses from the plants considered can hardly
be underestimated and needs to be explored further.
The total phenolic content in the leaf tissues studied varied according to the plant
species studied. Although no direct correlation was observed between larval
performance and the total polyphenols content in the leaf extracts, this does not
explicitly rule out the non-involvement of these compounds in larval host choice and
performance. Determination of plant total chemical substance does not allow
conclusive information on the importance of single component on a particular insect
behavioural trait. And in case of total polyphenols, past phytochemical and
experimental studies leave no doubt that individual phenolic compound varies
substantially with respect to biological activity (Bergvinson et al., 1994; Santiago et
al., 2005). Therefore, the effect of plant foliage phenolics on B. fusca larva may
depend on the specific chemical structure of the compound. Ascertaining the exact
117
role of each and specific phenolic compound on larval feeding responses will require
analysis of each phenolic compound and not of pooled contents in the plant leaves.
Study of digestive enzymes in herbivorous insects is not only important for the
understanding of digestion biochemistry but also for developing of safe and useful
control pest management strategies. Similar to other oligophagous insects, B. fusca
larvae appear to be dependent on digestive glycosidases (glucosidases and
galactosidases) than on protein/amino acid degrading enzymes (proteases) for
survival and performance. Therefore by considering the importance of carbohydrate
digestion as a target for B. fusca control, a study on their digestive enzymes could
definitely be crucial in adopting new control procedures. Additionally these
enzymes may be important in the degradation of secondary plant metabolites present
in B. fusca host plants and therefore important in the understanding of insect
herbivore coevolution. For example, high levels of C4 esterases induced after larvae
fed on high phenolic containing plants (S. megaphylla, P. deustum and P. maximum)
used in this study could be directly correlated with high levels of phenolic
compounds in the specific plant tissue. Clearly further biochemical and molecular
biological analysis are needed to fully understand the effect of such compounds on
the digestive physiology of B. fusca larva. Similarly, the mechanisms by which B.
fusca larvae is able to detoxify harmful allelochemicals or physiologically adapt to
host plants containing such toxic chemicals as the larvae and exploits diversifies to
variety of host plants needs to be further elucidated.
The refuges constituted by non-crop host plants may be useful in the integrated pest
management strategy as has been applied in the Push-pull system (Khan et al.,
2000). However the benefits of such refuges may be limited because in some cases
118
host plant diversity may drive genetic divergence and possibly even host-mediated
sympatric speciation (Mopper et al., 1996). However, in this study no host-plant
related genetic diversity was visible between populations sampled from Z. mays and
those sampled from A. donax as has been hypothesised (Senzolin, 2006). Moreover
the diversity in the genetic structure exhibited between the two populations may be
congruent with patterns of geographical differentiation supporting the findings of
Senzolin, (2006). However because of the limited samples and the not so efficient
methods of sampling used, it is premature to rule out the existence of host associated
populations of this species. Better and targeted methods thus need to be developed,
such as sampling in the same locality, time frame and on different plant species to
conclusively determine the existence of any genetic differentiation in respect to host
use among B. fusca populations.
In summary, the current study illustrates that an understanding of the specific
allelochemicals and plant physical characteristics perceived by insect sensory
system in a plant–insect system can give cues as to the type of physiological,
biochemical and genetic adaptations that can be exhibited by the insect. Therefore,
for sustainable management of B. fusca an integrated approach based on the
knowledge of important plant cues as well as physiological and genetic adaptations
involved in host selection and establishment needs to be adopted to radically
influence management initiatives of B. fusca.
119
6. 0: REFERENCES
Agrawal A.A., F. Vala and M.W. Sabelis, (2002). Induction of preference and
performance after acclimation to novel hosts in a phytophagous spider mite;
adaptive plasticity? The American Naturalists 159 (5): 554-565.
Ahmad S., L.B. Brattsen, C.A. Mullin and S.J. Yu, (1986). Enzymes involved in the
metabolism of plant allelochemicals, pp 73-152, in L.B. Brattsen and S. Ahmad
(Eds.), Molecular aspects of Insect-plant Associations, Plenum Press, New York, USA.
Akhtar Y. and M.B. Isman, (2003). Larval exposure to oviposition deterrents alters
subsequent oviposition behaviour in generalist Trichoplusia ni and specialist,
Plutella xylostella moths. Journal of Chemical Ecology 29: 1853-1870.
Albert P.J., (2003). Electrophysiological responses to sucrose from a gustatory
sensillum on the larval maxillary palp of the spruce budworm, Choristoneura
fumiferana (Clem.) (Lepidoptera: Tortricidae). Journal of Insect Physiology 49:
733-738.
Albert P.J., C. Cearly, F.E. Hanson and S. Parisella, (1982). Behavioural responses
of eastern spruce budworm larvae to sucrose and other carbohydrates. Journal of
Chemical Ecology 8: 233-239.
Anderson P., M. Hilker and J. Löfqvist, (1995). Larval diet influence on oviposition
behaviour in Spodoptera littoralis. Entomologia Experimentalis et Applicata 74: 71-82.
120
Ballebeni P. and M. Rahier, (2000). A quantitative genetic analysis of leaf beetle
larval performance on two natural hosts: including a mixed diet. Journal of
Evolutionary Biology 13: 98-106.
Barron A.B., (2001). The life and death of Hopkins’host-selection principle. Journal
of Insect Behaviour 14: 725–737.
Bartlet E., D. Parsons, I.H. Williams and S.J. Clark, (1994). The influence of
glucosinolates and sugars on feeding by the cabbage stem flea beetle, Psylliodes
chrysocephala. Entomologia Experimentalis et Applicata 73: 77-83.
Becerra J.X., (1997). Insects on Plants: Macroevolutionary chemical trends in host
use. Science 276: 253-256.
Becerra J.X. and E. Venable, (1999). Macroevolution of insect-plant associations:
The relevance of host biogeography to host affiliation. Proceedings of the National
Academy of Sciences of the United States of America 96: 12626-12631.
Berenbaum M., (1990). Evolution of specialization in insect-umbellifer associations.
Annual Review of Entomology 35: 319–343.
Berenbaum M., (1996). Introduction to the symposium: on the evolution of
specialisation. American Naturalists, 148 (Suppl): S78-S83.
121
Bergvinson D.J., (1993). Role of phenolic acids in maize resistance to the European
corn borer, Ostrinia nubilalis (Hubner). PhD dissertation. University of Ottawa,
Ontario, Canada.
Bergvinson D.J., J.J. Arnason and L.N. Pietrzak, (1994). Localisation and
quantification of cell wall phenolics in European corn borer resistant and susceptible
maize inbreds. Canadian Journal Botany 72: 1243-1249.
Berlocher S.H. and J.L. Feder, (2002). Sympatric speciation in phytophagous
insects: moving beyond controversy? Annual Review of Entomology 47: 773-815.
Bernays E.A., (2001). Neural limitations in phytophagous insects: Implications for
diet breadth and evolution of host affiliation. Annual Review of Entomology 46: 703-727.
Bernays E.A. (1991) Evolution of insect morphology in relation to plants.
Philosophical Transactions of the Royal Society of London B 333: 257–264.
Bernays E.A. and R.F. Chapman, (1994). Host-plant selection by phytophagous
insects. Chapman and Hall, New York, USA.
Bernays E.A. and W. Wcislo, (1994). Sensory capabilities, information processing
and resource specialization. Quaterly Review of Biology 69: 187-204.
122
Bernays E.A., D.J. Funk, and N.A. Moran, (2000). Intraspecific differences in
olfactory sensilla in relation to diet breadth in Uroleucon ambrosiae (Homoptera:
Aphididae). Journal of Morphology 245: 99–109.
Birkett M.A, K. Chamberlain, Z.R. Khan, J. A. Picket, T. Toshova, L.J. Wadhams.
C.M. Woodcook, (2006). Electrophysiological responses of the lepidopterous
stemborers Chilo partellus and Busseola fusca to volatiles from wild and cultivated
host plants. Journal of Chemical Ecology, 32: 2475-2487
Blackiston D.J., Casey E.S. and M.R. Weiss, (2008). Retention of memory through
metamorphosis: can moth remember what it learned as a caterpillar? PlosOne 3:1-7.
Bowdan E., (1995). The effects of a phagostimulant and a deterrent on the
microstructure of feeding by Manduca sexta caterpillars. Entomologia
Experimentalis et Applicata 77: 297-306.
Brattsten L.B., (1988). Enzymic adaptations in leaf-feeding insects to host-plant
allelochemicals. Journal of Chemical Ecology 14: 1919-1939.
Brattsten L.B., (1991). Bioengineering of crop plants and resistant biotype evolution
in insect. Archives of Insect Biochemistry and Physiology 17: 253-267.
Bruce T.J.A., L.J. Wadhams and C.M. Woodcock, (2005). Insect host location: a
volatile situation. Trends in Plant Science 10: 269-274.
123
Bush G.L., (1994). Sympatric speciation in animals: new wine in old bottles. Trends
in Ecological & Evolution 9: 285–288.
Cai C.Y., Y. Konno and K. Matsuda, (2002). Studies on the ovipositional
preferences in Helicoverpa assulta and Helicoverpa armigera. Tohoku Journal of
Agricultural Research 53: 11-23.
Calatayud P.-A, M. Chimtawi, D. Tauban, F. Marion-Poll, B.P. Le Rü, J.-F. Silvain,
and B. Frérot, (2006). Sexual dimorphism of antennal, tarsal and ovipositor
chemosensilla in the African stem borer, Busseola fusca (Fuller) (Lepidoptera:
Noctuidae). Annales de la Société Entomologique de France 42 (3-4): 403-412.
Calatayud P.-A., G. Juma, P.G.N. Njagi, N. Faure, S. Calatayud, S. Dupas, B. Le
Rü, G. Magoma, J.-F. Silvain and B. Frérot, (2008). Differences in mate acceptance
and host plant recognition between wild and laboratory-reared Busseola fusca
(Fuller). Journal of Applied Entomology 132: 255-264.
Camara M., (1997). A recent host range expansion in Junonia coenia
(Nymphalidae). Oviposition preference, survival, growth, and chemical defense.
Evolution 51: 873–884.
124
Campos F., J. Artkinson, J.T. Arnason, B.J.R. Philogene, P. Morand, N.H. Werstiuk
and G. Timmins, (1989). Toxicokinetics of 2, 4-dihydroxy-7-methoxy-1, 4-
benzoxazin-3-one (DIMBOA) in the European corn borer, Ostrinia nubilalis
(Hübner). Journal of Chemical Ecology 15: 1989-2001.
Carroll S.P.and C.Boyd, (1992). Host race radiation in the soapberry bug: natural
history with the history. Evolution 46: 1052–1069
Chérif M., A. Asselin and R.R. Bélanger, (1994). Defense responses induced by soluble
silicon in cucumber roots infected by Pythium spp. Phytopathology 84: 236-242.
Clausen T.P., J.W. Keller and P.B. Richardt, (1990). Aglycone fragmentation
accompanies β- glucosidase catalysed hydrolysis of Salicortin, a naturally occurring
phenol glycoside. Tetrahedron Letters 31: 4537-4538.
Colwell R.K, (1986). Population structure and sexual selection for host fidelity in
the speciation of hummingbird mites, pp: 475-495 in S. Karlin and E. Nevo (Eds.),
Evolutionary processes and theory, Academic press, New York, USA.
Conn E.E., (1980). Cyanogenic glycosides, pp. 461-492, in E.A. Bell and B.V.
Charlwood (Eds.), Secondary Plant Products, Springer-Verlag, Berlin, Heidelberg,
New York.
125
Cook S.M., Z.R. Khan and J.A. Pickett, (2007). The use of ‘‘push-pull’’ strategies in
integrated pest management. Annual Review of Entomology 52: 375-400.
Crespi B.J. and C.P. Sandoval, (2000). Phylogenetic evidence for the evolution of
ecological specialization in Timema walking sticks. Journal of Evolutionary Biology.
13: 249-262.
Cunningham J.P., M.F.A. Jallow, D.J. Wright and M.P. Zalucki, (1998). Learning in
host selection in Helicoverpa armigera (Hübner) (Lepidoptera:Noctuidae). Animal
Behaviour 55: 227–234.
De Boer G., (1993). Plasticity in food preference and diet-induced differential
weighting of chemosensory information in larval Manduca sexta. Journal of Insect
Physiology 39(1): 17-24.
De Boer G. and F.E. Hanson, (1984). Foodplant selection and induction of feeding
preference among host and non-host plants in larvae of tobacco hornworm Manduca
sexta. Entomologia Experimentalis et Applicata 35: 177-193.
DeGroote H., (2002). Maize yield losses from stemborers in Kenya. Insect science
and its Application 22: 89-96.
126
del Campo M.L. and J.A.A. Renwick, (2000). Induction of host specificity in larvae
of Manduca sexta: chemical dependence controlling host recognition and
developmental rate. Chemoecology 10: 115 -121.
del Campo C.I., M.L., Miles, F.C. Schroeder, C. Mueller, R. Booker and J.A.A.
Renwick, (2001). Host recognition by the tobacco hornworm is mediated by a host
plant compound. Nature 411: 186-89.
del Campo M.L. and C.I. Miles, (2003). Chemosensory tuning to a host recognition
cue in the facultative specialist larvae of the moth Manduca sexta. Journal of
Experimental Biology 206: 3979-3990.
Denno R.F., S. Larsson and K.L. Olmstead, (1990). Role of enemy free space and
plant quality in host-plant selection by willow beetles. Ecology 71: 124-137.
Denno R.F., M.S. McClure and J.R. Ott, (1995). Interspecific interactions in
phytophagous insects: Competition re-examined and resurrected. Annual Review of
Entomology 40: 297-331.
Diehl S.R. and Bush G.L. (1989). The role of habitat preference in adaptation and
speciation, pp. 527–553 in Otte D. and J.A. Endler (eds), Speciation and its
Consequences, Sinauer Associates, Sunderland, MA.
127
Dethier V.G., (1937). Gustation and olfaction in lepidopterous larvae. Biological
Bulletin 72: 7-23.
Dethier V.G., (1941). The function of the antennal receptors in lepidopterous larvae.
Biological Bulletin 80: 403-414.
Dethier V.G. and L.M. Schoonhoven, (1969). Olfactory coding by lepidopterous
larvae. Entomologia Experimentalis et Applicata 12: 535-543.
Dethier V.G. and J.H. Kuch, (1971). Electrophysiological studies of gustation in
lepidopterous larvae. I. Comparative sensitivity to sugars, amino acids and
glycosides. Zeitschrift Fuer Vergleichende Physiologie 72: 343-363.
Dethier V.G., (1980). Responses of some olfactory receptors on the eastern tent
caterpillar (Malacosoma americanum) to leaves. Journal of Chemical Ecology 6:
213-220.
Devitt B.D. and J.J.B. Smith, (1982). Morphology and fine structure of mouthpart
sensilla in the dark-sided cutworm Euxoa messoria (Harris) (Lepidoptera:
Noctuidae). International Journal of Insect Morphology and Embryology 11: 255-
270.
128
Djamin A. and M.D. Pathak, (1967). Role of silica in resistance to Asiatic rice borer,
Chilo suppressalis Walker, in rice varieties. Journal of Economic Entomology 60:
347–351.
Dobler S., P. Mardulyn, J.M. Pasteels and M. Rowell-Rahier, (1996). Host-plant
switches and the evolution of chemical defenses and life history in the leaf beetle
genus Oreina. Evolution 50: 2373-2386.
Drès M. and J. Mallet, (2002). Host races in plant-feeding insects and their
importance in sympatric speciation. Philosophical Transactions of the Royal
Society of London B Biological Sciences 357: 471–492
Duffey S.S. and M.J. Stout, (1996). Antinutritive and toxic components of plant
defense gainst insects. Archives of insect Biochemistry and Physiology 32: 3–37.
Dusenbery D.B., (1992). Sensory ecology. W.H. Freeman and Company, New York,
USA.
Dyer L.A., (1995). Tasty generalists and nasty specialists? Antipredator mechanisms
in tropical lepidopteran larvae. Ecology 76: 1483-1496.
Egas M. and M.W. Sabelis, (2001). Adaptive learning of host preference in an
herbivorous arthropod. Ecology Letters 4: 190–195.
129
Ehrlich P.R. and P.H. Raven, (1964). Butterflies and plants: A study in coevolution.
Evolution 18: 586-608.
Emelianov I., J. Mallet and W. Baltensweiler, (1995). Genetic differentiation in
Zeiraphera diniana (Lepidoptera: Tortricidae: the larch budmoth): polymorphism,
host races or sibling species. Heredity 75: 416-424.
Engler H.S., K.C. Spencer and L.E. Gilbert, (2000). Insect metabolism-preventing
cyanide release from leaves. Nature 406: 144-145.
Epstein E., (1999). Silicon. Annual Review of Plant Physiology and Plant Molecular
Biology 50: 641-664.
Faucheux M.J., (1995). Sensilla on the larval antennae and mouthparts of the
European sunflower moth, Homoeosoma nebulella Den. and Schiff. (Lepidoptera:
Pyralidae). International Journal of Insect Morphology and Embryology 24: 391-403.
Faucheux M.J., (1999). Biodiversité et unité des organes sensoriels des insectes
Lépidoptères. Bulletin de la Société des Sciences Naturelles de l’Ouest de la France
(Suppl. Hors Série): 294p.
Feder J.L., C.A. Chilcote and G. Bush, (1990). Regional local and microgeographic
allele frequency variation between apple and hawthorn populations of Rhagolettis
pomonella in western Michigan. Evolution 44: 595-608.
130
Felton G.W., C.B. Summers and A.J. Mueller, (1994). Oxidative responses in
soybean foliage to herbivory by bean leaf beetle and three cornered alfalfa hopper.
Journal of Chemical Ecology 20: 639-650.
Feyereisen R., (1999). Insect P450 enzymes. Annual Review of Entomology 44: 507-533.
Flowers R.W. and R.T. Yamamoto, (1992). Feeding on non-host plants by partially
maxillectomized tobacco hornworms (Manduca sexta: Lepidoptera: Sphyngidae).
Florida Entomologist 75: 89 -93.
Forister M.L., (2005). Independent inheritance of preference and performance in
hybrids between host races of Mitoura butterflies (Lepidoptera: Lycaenidae).
Evolution 59, 1149–1155.
Fox C.W., (1993). A quantitative genetic analysis of oviposition preference and
larval performance on two hosts in the bruchid beetle Callosobruchus maculatus.
Evolution 47: 166–175.
Fox C.W. and R.G. Lalonde, (1993). Host confusion and the evolution of insect diet
breadths. Oikos 67: 577-581.
Frenzel M. and R. Brandl, (1998). Diversity and composition of phytophagous
insect guilds on Brassicaceae. Oecologia 113: 391–399.
131
Futuyma D.J and G. Moreno, (1988). The evolution of ecological specialization.
Annual Review of Ecological Systematics 19: 207–233.
Futuyma D.J., (1999). Potential evolution of host range in herbivorous insects.
Proceedings: Host specificity testing of exotic arthropod biological control agents:
The biological basis for improvement in safety. pp: 42-53.
Gaston K.J., D. Reavey and G.R. Valladares, (1992). Intimacy and fidelity: internal
and external feeding by the British Microlepidoptera. Ecological Entomology 17: 86-88.
Gassmann A.J., A. Levy, T. Tran and D.J. Futuyma, (2006). Adaptations of an
insect to a novel host plant: a phylogenetic approach. Functional Ecology 20: 478-485.
Glendinning J.I. and N.A. Gonzalez, (1995). Gustatory habituation to deterrent
allelochemicals in a herbivore-concentration and compound specificity. Animal
Behaviour 50: 915-927.
Glendinning J.I, S. Valcic and B.N. Timmermann, (1998). Maxillary palps can
mediate taste rejection of plant allelochemicals by caterpillars. Journal of
Comparative Physiology 183: 35-44.
Gomes F.B., J.C. Moraes, C.D dos Santos and M.M. Goussain, (2005). Resistance
induction in wheat plants by silicon and aphids. Scientia Agricola 62: 547–551.
132
Gomez L., E. Rubio and M. Augé, (2002). A new procedure for extraction and
measurement of soluble sugars in ligneous plants. Journal of the Science of Food
and Agriculture. 82: 360-369.
Gomez-Zurita J., C. Juan and E. Petitpierre, (2000). The evolutionary history of the
genus Timarcha (Coleoptera, chyrsomelidae) inferred from mitochondrial COII
gene and partial 16S rDNA sequences. Molecular Phylogenetics and Evolution 14:
304-317.
Goodman R.N., Z. Kiraly and K.R. Wood, (1986). Secondary metabolite, pp. 211-
224, in Goodman R.N. (Ed.), The Biochemistry and Physiology of Plant Disease.
Missouri, University of Missouri, USA.
Gould, F., (1998). Sustainability of transgenic insecticidal cultivars: Integrating pest
genetics and ecology. Annual Review of Entomology 43: 701-726.
Grimes L.R. and H.H. Neunzig, (1986). Morphological survey of the maxillae in last
stage larvae of the suborder Ditrysia (Lepidoptera). Mesal lobes (Laciniogaleae).
Annals of the Entomological Society of America 79: 510-526.
Groman, J. D. and O. Pellmyr, (2000). Rapid evolution and evolution and
specialisation following host colonization in a yucca moth. Journal of Evolutionary
Biology 13: 223-236.
133
Gupta P.D and A.J. Thorsteinson, (1960). Food plant relationships of diamond-back
moth Plutella maculipennis (curt.). I. Gustation and olfaction in relation to botanical
specificity of the larvae. Entomologia Experimentalis et Applicata 3: 241-250.
Haile A. and T. Hofsvang, (2002). Host plant preference of the stem borer Busseola
fusca (Fuller) (Lepidoptera: Noctuidae). Crop Protection 21: 227–233.
Hammerschmidt R., (2005). Silicon and plant defense: the evidence continues to
mount. Physiological and Molecular Plant Pathology 66: 117-118.
Harris K.M. and K.F. Nwanze, (1992). Busseola fusca (Fuller), the African maize
stem borer; a handbook of information. Information Bulletin No 33, ICRISAT, CABI,
Oxon, UK.
Harry M., M. Solignac and D. Lachaise, (1998). Molecular evidence for parallel
evolution of adaptive syndromes in fig-breeding Lissocephala (Drosophilidae).
Molecular Phylogenetics and Evolution 9: 542–551.
Haukioja E., P. Niemala and S. Siren, (1985). Foliage phenols and Nitrogen in
relation to growth, insect damage, and ability to recover the defoliation, in the
mountain birch, Betula pubescens ssp. tortosa. Oecologia 65: 214-222.
134
Hemming J.D.C. and R. L. Lindroth, (1999). Effects of light and Nuetrient
availability on aspen: growth, phytochemistry and insect performance. Journal of
Chemical Ecology 25 1687-1714.
Hemming J.D.C. and R. L. Lindroth, (2000). Effects of phenolic glycosides and
protein on Gypsy Moth ( Lepidoptera: Lymantriidae) and Forest tent caterpillar
(Lepidoptera:Lasiocampidae) performance and detoxification activities.
Environmental Entomology 29: 1108-1115.
Herms D.A. and W.J. Mattson, (1992). The dilemma of plants to grow or defend.
Quarterly Review of Biology 67: 283-335.
Hinks C.F. and M.A. Erlandson, (1994). The accumulation of haemolymph proteins
and activity of digestive proteinases of grasshoppers (Melanoplus sanguinipes) fed
wheat, oats or kochia. Journal of Insect Physiology 41: 425-433.
Hodgson E. S., J.Y. Lettvin, K.D. Roeder, (1955). Physiology of a primary
chemoreceptor unit. Science 122: 417-418.
Honda K., (1995). Chemical basis of differential oviposition by lepidopterous
insects. Archives of Insect Biochemistry and Physiology 30: 1-23.
Hopkins A.D., (1917). A description of G.C. Hewitt’s paper on “Insect behavior as a
factor in applied entomology”. Journal of Economic Entomology 10: 92-93.
135
Huang X.P. and J.A.A. Renwick, (1995). Cross habituation to feeding deterrents
acceptance of a marginal host plant by Pieris rapae larvae. Entomologia
Experimentalis et Applicata 76: 295-302.
Huang X.P. and J.A.A. Renwick, (1997). Feeding deterrents and sensitivity
suppressors for Pieris rapae larvae in wheat germ diet. Journal of Chemical Ecology
23: 51-70.
Hudson R.R, M. Slatkin and W.P. Maddison, (1992). Estimation of levels of gene
flow from DNA sequence data. Genetics 132: 583–589.
Hwang S.Y. and R.L. Lindroth, (1997). Clonal variation in foliar chemistry of
Aspen: Effects on gypsy moths and forest tent caterpillars. Oecologia 111: 99-108.
Ikonen A., M. Sipura, S. Miettinen and J. Tahvanainen, (2003). Evidence of host
race formation in the leaf beetle Galerucella lineola. Entomologia Experimentalis et
Applicata 108: 179-185.
Ingram W.R., (1958). The lepidopterous stalk-borers associated with Graminae in
Uganda. Bulletin of Entomological Research 40: 367-383.
Ishikawa S., T. Hirao and N. Arai, (1969). Chemosensory basis of host plant
selection in the silkworm. Entomologia Experimentalis et Applicata, 12: 544-554.
136
Ishikawa S., (1963). Response of maxillary chemoreceptors in the larva of the
silkworm Bombyx mori to stimulation of carbohydrates. Journal of Cellular and
Comparative Physiology 61: 99-107.
Jaenike J., (1988). Effects of early adult experience on host selection in insects:
some experimental and theoretical results. Journal of Insect Behavior 1: 3-15.
Jaenike J., (1990). Host specialization in phytophagous insects. Annual Review of
Ecology and Systematics 21: 243–273.
Janz N. and S. Nylin, (1998). Butterflies and plants: a phylogenetic study. Evolution
52: 486–502.
Janz N., K. Nyblom and S. Nylin, (2001). Evolutionary dynamics of host-plant
specialization: a case study of the tribe Nymphalini. Evolution 55: 783–796.
Janz N., Nylin S. and N. Wahlberg, (2006). Diversity begets diversity: host
expansions and the diversification of plant-feeding insects. BMC Evolutionary
Biology 6: 4.
Janz, N. and S. Nylin, (2008) "The oscillation hypothesis of host plant-range and
speciation". In K. J. Tilmon (ed.), "Specialization, Speciation, and Radiation: the
Evolutionary Biology of Herbivorous Insects". pp. 203-215. University of California
Press, Berkeley, California.
137
Jarvis S.C. and L.H.P. Jones, (1987). The absorption and transport of manganese by
perennial ryegrass and white clover as affected by silicon. Plant soil 99: 231-240.
Jermy T., F.E. Hanson and V.G. Dethier, (1968). Induction of specific food
preference in lepidopterous larvae. Entomologia Experimentalis et Applicata 11:
211-230.
Jukes T.H. and C.R. Cantor, (1969). Evolution of protein molecules, pp. 21–132 in
Munro H.N. (Ed.), Mammalian Protein Metabolism,. Academic Press, New York,
NY, USA.
Juma G., (2005). Role of plants volatile and surface metabolites in mediating host
selection and oviposition of Busseola fusca (Fuller) (Lepidoptera: Noctuidae).
Master of Science (Biochemistry) of the Jomo Kenyatta University of Agriculture
and Technology, Kenya.
Kaufmann T., (1983). Behavioural biology, feeding habits and ecology of three
species of maize stemborers: Eldana saccharina (Lepidoptera: Pyralidae), Sesamia
calamistis and Busseola fusca (Noctuidae) in Ibadan, Nigeria, West Africa. Journal
of the Georgia Entomological Society 18: 259-272.
Kawecki T.J., (1994). Accumulation of deleterious mutations and the evolutionary
cost of being generalists. American Naturalists 144: 833-838.
138
Kawecki, T.J., (1997). Sympatric speciation via habitat specialisation driven by
deleterious mutations. Evolution 51: 1751-1763.
Keeping M.G. and J.H. Meyer, (2002). Calcium silicate enhances resistance of
sugarcane to the African stalk borer Eldana saccharina Walker (Lepidoptera:
Pyralidae). Agricultural and Forest Entomology 4: 265–274.
Keeping M.G. and J.H. Meyer, (2006). Silicon-mediated resistance of sugarcane to
Eldana saccharina Walker (Lepidoptera: Pyralidae): effects of silicon source and
cultivar. Journal of Applied Entomology 130: 410–420.
Keeping M.G. and O.L. Kvedaras, (2008). Silicon as a plant defense against insect
herbivory: response to Massey, Ennos and Hartley. Journal of Animal Ecology 77:
631-633.
Kfir R., W.A. Overholt, Z.R. Khan and A. Polaszek, (2002). Biology and
management of economically important lepidopteran cereal stem borers in Africa.
Annual Review of Entomology 47: 701-731.
Khan, Z. R. and J.A. Pickett, (2004). The ‘push-pull’ strategy for stemborer
management : A case study in exploiting biodervesity and chemical ecology, pp,
155-164, in G.M Gurr, S.D. Wratten, and M.A Altieri (eds). Ecological Engineering
for Pest Management. CABI, Oxford, UK.
139
Khan L.R., J.A. Litsinger, A.T. Barrion, F.F.D. Villanueva, N.J. Fernandez and L.D.
Taylo, (1991). World Bibliography of rice stemborer. International Rice Research
Institute, Los Banov, Phillipines and I.C.I.P.E, Nairobi.
Khan Z.R., J.A. Pickett, J. van den Berg, L.J. Wadhams and C.M. Woodcock,
(2000). Exploiting chemical ecology and species diversity: stem borer and striga
control for maize and sorghum in Africa. Pest Management Science 56: 957-962.
Kim K.C. and B.A. McPheron, (1993). Biology of variation; epilogue, pp. 453-468
in Kim K.C. and B.A. McPheron (Eds.) Evolution of insect pests, patterns of
variation, John Wiley, New York, USA.
Kvedaras O.L., M.G. Keeping, F.-R. Goebel and M. Byrne, (2005). Effects of
silicon on the African stalk borer, Eldana saccharina walker (Lepidoptera:
Pyralidae) in sugarcane. Proceeding of the South African Sugar Technologists
Association 79: 359-362.
Kvedaras O.L. and M.G. Keeping, (2007). Silicon impedes stalk penetration by the
borer Eldana saccharina in sugarcane. Entomologia Experimentalis et Applicata
125: 103–110.
140
Kvedaras O.L., M.G. Keeping, F.-R. Goebel and M.J. Byrne, (2007a). Water stress
augments silicon-mediated resistance of susceptible sugarcane cultivars to the stalk
borer, Eldana saccharina Walker (Lepidoptera: Pyralidae). Bulletin of
Entomological Research 97: 75-183.
Kvedaras O.L., M.G. Keeping, F.-R. Goebel and M.J. Byrne, (2007b). Larval
performance of the pyralid borer Eldana saccharina Walker and stalk damage in
sugarcane: influence of plant silicon, cultivar and feeding site. International Journal
of Pest Management 53: 183–194.
Laing M.D., M.C. Gatarayiha and A. Adandonon, (2006). Silicon use for pest
control in agriculture: a review. Proceedings of the South African Sugar
Technologists' Association, 80: 278–286.
Langor D.W. and J.R. Spence, (1991). Host effects on allozyme and morphological
variation of the mountain pine beetle, Dendroctonus ponderosae Hopkins
(Coleoptera, Scolytidae). Canadian Entomologist 123, 395–410.
Lazarevic J. and V. Peri-Mataruga,(2003). Nutritive stress effects on growth and
digestive physiology of Lymantria dispar.Yugoslavia Medical Biochemistry22:53-59.
Larsson S. and B. Ekbom, (1995). Oviposition mistakes in herbivorous insects:
confusion or a step towards a new host plant? Oikos 72: 155-160.
141
Lee K.P., D. Raubenheimer, S.T. Behmer and S.J. Simpson, (2003). A correlation
between macronutrient balancing and insect host-plant range: evidence from the
specialist caterpillar Spodoptera exempta (Walker). Journal of Insect Physiology 49:
1161-1171.
Le Ru B.P., G.O. Ong’amo, P. Moyal, E. Muchugu, L. Ngala, B. Musyoka, Z.
Abdullah, T. Matama-Kauma, V.Y. Lada, B. Pallangyo, C.O. Omwega, F.
Schulthess, P.-A. Calatayud and J.-F. Silvain, (2006a). Geographic distribution and
host plant ranges of East African noctuid stem borers. Annales de la Société
Entomologique de France 42: 353-361.
Le Ru B.P., G.O. Ong’amo, P. Moyal, L. Ngala, B. Musyoka, Z. Abdullah, D.
Cugala, B. Defabachew, T.A. Haile, T. Matama-Kauma, V.Y. Lada, B. Negassi, B.
Pallangyo, J. Ravololonandrianina, A. Sidumo, C.O. Omwega, F. Schulthess, P.-A.
Calatayud and J.-F. Silvain, (2006b). Diversity of lepidopteran stemborers on
monocotyledonous plants in eastern Africa and island of Madagascar and Zanzibar
revisited. Bulletin of Entomological Research 96: 1-9.
Leniaud L., P. Audiot, D. Bourguet, B. Frérot, G. Genestier, S. F.Lee, T.Malausa,
A.H. Le Pallec, M.C. Souqual and S. Ponsard, (2006). Genetic structure of European
and Mediterranean maize borer populations on several wild and cultivated host
plants. Entomologia Experimentalis et Applicata 120: 51-62.
142
Lindroth R.L., (1989). Chemical ecology of luna moth: effects of host plant on
detoxification enzyme activity. Journal of Chemical Ecology 15: 2019-2029.
Lindroth R.L. and J.D.C. Hemming, (1990). Responses of the gypsy moth
(Lepidoptera: Lymantriidae to tremulacin, an aspen phenolic glycoside.
Environmental Entomology 19: 842-847.
Lindroth R.l. and M.S. Bloomer, (1991). Biochemical ecology of the forest tent
caterpillar: Response to dietary proteins and phenolic glycosides. Oecologia 86:
408-413.
Lindroth R.L. and A.V. Weisbrod, (1991). Genetic variation in response of the
gypsy moth to aspen phenolic glycosides. Biochemical Systematic Ecology 19: 97-103.
Lindroth R.L., S.M. Jung, and A.M. Feuker, (1993). Detoxification activity in the
gypsy moth: effects of host CO2 and NO3- availability. Journal of Chemical Ecology
19: 357-367.
Lopez J.D. Jr. and P.D. Lingren, (1994). Feeding response of adult Helicoverpa zea
(Lepidoptera: Noctuidae) to commercial phagostimulants. Journal of Economic
Entomology 87: 1653-1658.
Lynch M. and T.J. Crease, (1990). The analysis of population survey data on DNA
sequence variation. Molecular Biology and Evolution 7: 377–394.
143
Ma, J.F., (2004). Role of silicon in enhancing the resistance of plants to biotic and
abiotic stresses. Soil Science and Plant Nutrition 50: 11–18.
Ma W. C., (1976). Mouth parts and receptors involved in feeding behaviour and
sugar perception in the African armyworm, Spodoptera exempta (Lepidoptera,
Noctuidae. Symp. Biol. Hung. 16: 139-151.
Ma J.F. and E. Takahashi, (2002) (Eds.). Soil, fertilizer, and Plant silicon research
in Japan. Elsevier Science, Amsterdam, The Netherlands.
Ma J.F. and N. Yamuji, (2006). Silicon uptake and accumulation in higher plants.
Trends in Plant Science 11: 392-397.
Ma W.-C. and I. Kubo, (1977). Phagostimulants for Spodoptera exempta:
Identification of adenosine from Zea mays. Entomologia Experimentalis et
Applicata 2: 107-112.
Maddison D.R. and W.P. Maddison, (2001). MacClade. Sinauer, Sunderland, MA,
USA.
Mac-Arthur R.H. and R. Levins, (1964). Competition, habitat selection and
character displacement in a patchy environment. Proceeding of the National
Academy of Science of the United States of America 51: 1207-1210.
144
Margaritopoulos, J.T., J.A. Tsitsipis, E.Zintzaras, and R.L.Blackman, (2000). Host-
correlated morphological variation of Myzus persicae (Hemiptera: Aphididae)
populations in Greece. Bulletin of Entomological Research 90: 233–244.
Martel C., A. Rejasse, F. Rousset, M.-T. Bethenod and D. Bourguet, (2003). Host
plant associated genetic differentiation in the northern French populations of the
European corn borer. Heredity 90: 141-149.
Massey F.P., A.R. Ennos and S.E. Hartley, (2006). Silica in grasses as a defence
against insect herbivores: contrasting effects on folivores and a phloem feeder.
Journal of Animal Ecology 75: 595–603.
Mendiola-Olaya E., A. Valencia-Jimenez, S. Valdes-Roddriguez, J. Delano-Frier
and A. Blanco-Labra, (2000). Digestive amylase from the larger grain borer,
Prostephanus truncates Horn,. Comparative Biochemical Physiology. Part B 126:
425-433.
Meyer J.H. and M.G. Keeping, (2005). Impact of silicon in alleviating biotic stress
in sugarcane in South Africa. Sugarcane International 23: 14-18.
Miller J.R. and R.S. Cowles, (1990). Stimulo-deterrent diversion: a concept and its
possible application to onion maggot control. Journal of Chemical Ecology 16 :
3197-3212.
145
Mitter C. and B.D. Farrell, (1991). Macroevolutionary aspects of insect-plant
relationships, pp. 35-78, in E. Bernays (Ed.), Insect/Plant Interactions, vol. 3., CRC
Press, Boca Raton, USA.
Mohamed M.A., S.S. Quisenberry and D.J. Moellenbeck, (1992). 6, 10, 14-
trimethylpentadecan-2-one: a Bermuda grass phagostimulant to fall armyworm
(Lepidoptera: Noctuidae). Journal of Chemical Ecology 18 (4): 673-682.
Mopper S. (1996). Adaptive genetic structure in phytophagous insect populations.
Trends in Ecology & Evolution 11: 235-238.
Nayak S. and R.N. Singh, (1983). Sensilla on the tarsal segments and mouthparts of
adult Drosophila melanogaster Meigen (Diptera: Drosophilidae). International
Journal of Insect Morphology and Embryology 12: 273-291.
Nei M., (1987). Molecular Evolutionary Genetics. Columbia University Press, New
York, NY, USA.
Niemeyer H.M., (1988). Hydroxamic acids (4-hydroxy-1,4-benzoxazin-3-ones),
defence chemicals in the Gramineae. Phytochemistry 27: 3349-3358.
Nylin S. and N. Janz, (1999). Ecology and evolution of host plant range: butterflies
as a model group, pp. 31–54 in H. Olff, V.K. Brown and R.H. Drent (Eds.),
Herbivores: between plants and predators, Blackwell Science, Oxford, UK.
146
Nylin S., A. Bergström and N. Janz, (2000). Butterfly host plant choice in the face
of possible confusion. Journal of Insect Behavior 13: 469-482.
Nylin S. and N. Wahlberg, (2008). Does plasticity drive speciation? Host plant shifts
and diversification in nymphaline butterflies (Lepidoptera: Nymphalidae) during the
tertiary. Biological Journal of the Linnean Society 94: 115-13.
Nwanze K.F., (1988). Distribution and seasonal incidence of some major pests of
sorghum in Burkina Faso. Insect Science and Its Application 9: 313-321.
Ojeda-Avila T., H.A. Woods and R.A. Raguso, (2003). Effects of dietary variation
on growth, composition and maturation of Manduca sexta (Sphingidae:
Lepidoptera). Journal of Insect Physiology 49: 293-306.
Ong’amo G.O., B. Le Rü, S. Dupas, P. Moyal, P.-A. Calatayud and J.-F. Silvain,
(2006). Distribution, pest status and agro-climatic preferences of lepidopteran stemborers
of maize in Kenya. Annales de la Société Entomologique de France 42:171-177.
Ong’amo G.O., B.P. Le Rü, S. Dupas, P. Moyal, E. Muchugu, P.-A. Calatayud and
J.-F. Silvain, (2006). The role of wild host plants in the abundance of lepidopteran
stem borers along altitudinal gradient in Kenya. Annales de la Société
Entomologique de France 42 (3-4) : 363-370.
147
Ong'amo G.O., B.P. Le Rü, P. Moyal, P.-A. Calatayud, P. Le Gall, C.K.P.O. Ogol,
E.D. Kokwaro, C. Capdevielle-Dulac and J.-F. Silvain, (2008). Host plant diversity
of Sesamia calamistis: cytochrome b gene sequences reveal local genetic
differentiation. Entomologia Experimentalis et Applicata 128: 154-161.
Onyango F.O. and J.P.R. Ochieng’-Odero, (1994). Continuous rearing of the maize
stem borer Busseola fusca on an artificial diet. Entomologia Experimentalis et
Applicata 73: 139-144.
Ortego, F., C. Novillo and P. Castanera, (1996).Characterization and distribution of
digestive proteases of the stalk corn borer, Sesamia nonagrioides Lef. (Lepidoptera:
Noctuidae). Archives of insect Biochemistry and Physiology 33: 163-180.
Ortego F., Ruiz, M. and P. Castanera, (1998). Effect of DIMBOA on growth and
digestive physiology of Sesamia nonagriodes (Lepidoptera: Noctuidae) larvae.
Journal of Insect Physiology 44: 95-101.
Panda N. and G.S. Kush, (1995) (Eds.). Host plant resistance to insects. CAB
International, Wallingford, UK.
Papaj D.R., (1986). Interpopulation differences in host preference and the evolution
of learning in the butterfly, Battus philenor. Evolution 40: 518–530.
148
Parmesan C., M.C. Singer and I. Harris, (1995). Absence of adaptive learning from
the oviposition foraging behavior of a checkerspot butterfly. Animal Behaviour 50:
161–175.
Pashley D.P., (1986). Host association genetic differentiation in fall armyworm
(Lepidoptera: Noctuidae): a sibling species complex? Annals of the Entomological
Society of America 79: 898-904.
Peri-Mataruga V., D. Blagojevi, M.B. Spasi, J. Ivanovi and M. Jankovi-Hladni,
(1997). Effect of the host plant on the antioxidative defense in the midgut of
Lymantria dispar L. caterpillars of different population origins. Journal of Insect
Physiology 43:101-106.
Peterson M.A. and R.F. Denno, (1998). The influence of dispersal and diet breadth
on patterns of genetic isolation by distance in phytophagous insects. American
Naturalists 152: 428-446.
Peterson S.S., J.M. Scriber and J.G. Coors, (1988). Silica, cellulose and their
interactive effects on the feeding performance of the southern armyworm
Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae). Journal of Kansas
Entomological Society 61: 169–177.
Pilson D. and M.D. Rausher, (1988). Clutch size adjustment by a swallowtail
butterfly. Nature 333: 361–363.
149
Polaszek A. and Z.R. Khan, (1998). Host Plants. In : A. Polaszek (Eds.). African
Cereal Stem Borers: Economic Importance, Taxonomy, Natural Enemies and
Control. Wallingford, UK: CABI: 3-10.
Powell J., C. Mitter and B.D. Farrell, (1998). Evolution of larval food preference in
Lepidoptera, pp. 403-422 in N.P. Kristensen (Ed.), Moths and Butterflies. Volume 1.
Evolution, Systematics, and Biogeography. Handbook of Zoology. Volume IV.
Arthropoda: Insecta, Walter deGruyter, Berlin, Germany.
Prowell D.P., M. McMichael and J.-F. Silvain, (2004). Multilocus genetic analysis
of host use, introgression, and speciation in host strains of fall armyworm
(Lepidoptera: Noctuidae). Annals of Entomological Society of America 97: 1034–1044.
Radtkey R.R. and M.C. Singer, (1995). Repeated reversals of host-preference
evolution in a specialist insect herbivore. Evolution 49: 351–359.
Rahbé Y., N. Sauvion, G. Febvay, W.J. Peumans and A.M.R. Gatehouse, (1995).
Toxicity of lectins and processing of ingested proteins in the pea aphid
Acyrthosiphon pisum. Entomologia Experimentalis et Applicata 76: 143-155.
Raupp M.J., (1985). Effects of leave toughness on mandibular wear of the leaf
beetle, Plagiodera versicolora. Ecological Entomology 10: 73-79.
150
Redmond C.T. and D.A. Potter (2007). Silicon fertilization does not enhance
creeping bentgrass resistance to cutworms and white grubs. USGA Turfgrass and
Environmental Research Online 6: 1–7.
Renwick J.A.A. and F.S. Chew, (1994). Oviposition behavior in lepidoptera. Annual
Review of Entomology 39: 377-400.
Rémus-Borel W., J.G. Menzies and R.R. Bélanger (2005). Silicon induces
antifungal compounds in powdery mildew-infected wheat. Physiological and
Molecular Plant Pathology 66: 108-115.
Rice W.R., (1987). Speciation via habitat specialization: the evaluation of
reproductive isolation as a correlated character. Evolutionary Ecology 1: 301-314.
Rietdorf K. and J.L.M. Steidle, (2002). Was Hopkins right? Influence of larval and
early adult experience on the olfactory response in the granary weevil Sitophilus
granarius (Coleoptera, Curculionidae). Physiological Entomology 27(3): 223-227
Ristanovic D., (2001). Maïs (Zea mays L.), pp 44-70 in Raemaekers R. H. (Ed.),
Agriculture en Afrique Tropicale, Direction Générale de la Coopération
Internationale, Bruxelle, Belgique.
151
Roessingh P., S. Xu and S.J. Menken, (2007). Olfactory receptors on the maxillary
palps of small ermine moth larvae: evolutionary history of benzaldehyde sensitivity.
Journal of Comparative Physiology A 193 (6): 635-647.
Rodrigues F.A., D.J. McNally, L.E. Datnoff, J.B. Jones, C. Labbé, N. Benhamou,
J.G. Menzies and R.R. Bélanger, (2004). Silicon enhances the accumulation of
diterpenoid phytoalexins in rice: a potential mechanism for blast resistance.
Phytopathology 94: 177–183.
Rosenthal G.A. and M.R. Berenbaum, (1991) (Eds.). Herbivores. Their interaction
with secondary plant metabolites. Vols I and II, 2nd ed., Academic press, New York, USA.
Rozas J., J.C. Sànchez-DelBarrio, X. Messeguer and R. Roza, (2003). DnaSP, DNA
polymorphism analyses by the coalescent and other methods. Bioinformatics 19:
2496–2497.
Saito K., A. Yamamoto, T. Sa and M. Saigusa, (2005). Rapid, micro-methods to
estimate plant silicon content by dilute Hydrofluoric acid extraction and
spectrometric molybdenum method. Soil Science and Plant Nutrition 51: 29-36.
Sangster A.G., M.J. Hodson and H.J. Tubb, (2001). Silicon deposition in higher
plants, pp. 85-113, in Datnoff L.E, Snyder G.H., Korndorfer G.H. (Eds.), Silicon in
Agriculture. Elsevier Science, Amsterdam, The Netherlands.
152
Santiago R., R.A. Malvar, M.D. Baamonde, P. Revilla and X.C. Souto, (2005). Free
phenols in maize pith and their relationship with resistance to Sesamia nonagrioides
(Lepidoptera: Noctuidae) attack. Journal of Economic Entomology 98: 1349-1356.
SAS Institute, (2003). Users' guide:Statistics, SAS Institute, Cary, NC, USA.
Scheck A.L. and F. Gould, (1993).The genetic basis of host range in Heliothis
Virescens:larval survival and growth. Entomologia Experimentalis et Applicata 69:157-172.
Seshu Reddy K.V and P.T. Walker, (1990). A review of the yield losses in
graminaceous crops caused by Chilo spp. Insect Science and its Application 11: 563-569.
Seshu Reddy K.V., (1991). Insect pest of sorghum in Africa. Insect Science and its
Application 12: 653-657.
Schneider S.D., D. Roessli and L. Excoffier, (2000). ARLEQUIN (version 2000): A
Software for Genetic Data Analysis. Genetics and Biometry Laboratory, University
of Geneva, Geneva, Switzerland.
Schoonhoven L.M. and V.G. Dethier, (1966). Sensory aspects of host plant
discrimination by lepidopterous larvae. Archives Néerlandaises de Zoologie 16: 497-530.
Schoonhoven L.M., (1967). Some cold receptors in larvae of three lepidopterous
species. Journal of Insect Physiology 13: 821-828.
153
Schoonhoven L.M, (1987). What makes a caterpillar to eat? The sensory code
underlaying feeding behaviour. In perspectives in chemoreception and behaviour
(ed. R.F. Chapman, E.A. Bernays and J.G. Stoffolono), pp. 69-97. New York:
Springer-verlag.
Schoonhoven L.M., T. Jermy and J.J.A. van Loon, (1998) (Eds.). Insect-Plant
Biology: From Physiology to Evolution. Chapman & Hall, London, UK.
Schoonhoven L.M., J.J.A. van Loon and M. Dicke, (2005) (Eds.). Insect plant
biology. Oxford University Press, Oxford, UK.
Sezonlin M., S. Dupas, B. Le Rü, P. Le Gall, P. Moyal, P.-A. Calatayud, I. Giffard,
N. Faure and J.-F. Silvain, (2006). Phylogeography and population genetics of the
maize stalk borer Busseola fusca (Lepidoptera, Noctuidae) in sub-Saharan Africa.
Molecular Ecology 15 (2): 407-420.
Sétamou M., F. Schulthess, N.A. Bosque-Perez and A. Thomas-Odjo, (1993). Effect
of plant nitrogen and silica on the bionomics of Sesamia calamistis (Lepidoptera:
Noctuidae). Bulletin of Entomological Research 83: 405-411.
Sharma H.C., (1994). Phagostimulant activity of sucrose, sterols and soyabean leaf
extractables to the cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae). Insect
Science and its Application 15: 281-286.
154
Shields V.D.C., (1994). Ultrastructure of the aporous sensilla on the galea of larval
Mamestra configurata (Walker) (Lepidoptera: Noctuidae). Canadian Journal of
Zoology 72: 2032-2054.
Simmons R.B. and S.J. Weller, (2001). Utility and evolution of cytochrome b in
insects. Molecular Phylogenetics & Evolution 20: 196-210.
Simon C., F. Frati, A. Beckenbach, B. Crespi, H. Liu and P. Flook, (1994).
Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and
a compilation of conserved PCR primers. Annals of the Entomological Society of
America 87: 651–701.
Singer M.C., C.D. Thomas, H.L.Billington C. Parmesan, (1989). Variation among
conspecific insect populations in the mechanistic basis of diet breadth. Animal
Behaviour 37: 751-759.
Singer M.C., D. Vasco, C. Parmesan, C.D. Thomas and D. Ng, (1992).
Distinguishing between ‘preference’ and ‘motivation’ in food choice: an example
from insect oviposition. Animal Behaviour 44: 463-471.
Singer M., C.D. Thomas and C. Parmesan, (1993). Rapid human-induced evolution
of insect diet. Nature 366: 681–683.
155
Sithole S.Z., (1989). Sorghum stem borers in southern Africa. In: Nwanze K.F.
(Eds). International workshop sorghum stem borers, Patancheru, 17-20 November
1987, Patancheru, India: ICRISAT, pp: 41-47.
Slansky F. Jr, (1992). Allelochemical nuetrientinteractions in herbivore nuitritional
ecology, pp. 135-174, in G.A. Rosenthal and M.R. Berenbaum (Eds.), Herbivores:
In the interactions with secondary Plant metabolites, 2nd edition, Vol. 1, The
chemical Participants, Academic Press, New York, USA.
Southwood T.R.E., (1972). The insect plant relationship: an evolutionary
perspective. Symposium of the Royal Entomological Society of London 6:3-30. 1972.
Städler E., (2002). Plant chemical cues important for egg deposition by herbivorous
insects, pp. 171-197 in Hilker M, and Meiners T (Eds.), Chemoecology of insect
eggs and egg deposition, Blackwell publishing, Berlin, Germany.
Steglich W., B. Fugmann and S. Lang-Fugman (2000) (Eds.). ROMPP
Encyclopedia, Natural products. Georg Thieme Verlag, Sttugart and New York.
Stoyenhof J.L, J.A. Witter and M.E. Montgomery, (1994). Effects of host switching
on gypsy moth ( Lymantria dispar (L) under field conditions. Oecologia 97:143-157.
156
Strong D.R., J.H. Lawton and R. Southwood, (1984) (Eds.). Insects on plants.
Community patterns and mechanisms. Harvard University Press, Cambridge,
Massachusetts, USA.
Tallamy D.W., (2000). Physiological issues in host range expansion, pp.11-26 in R.
Van Dreisch, T. Heard, A. McClay and R. Reardon (Eds.), Proceedings of Session:
Host-Specificity Testing of Exotic Arthropod Biological Control Agents – The
Biological Basis for Improvement in Safety.
Tams W.H.T. and J. Bowden, (1953). A revision of the African species of Sesamia
Guenee and related genera (Agrotidae-Lepidoptera). Bulletin of Entomological
Research 43: 645-678.
Thompson J.N., 1994 (Ed.). The co-evolutionary process. University of Chicago
Press, Chicago, USA.
Thompson J.N. and O. Pellmyr, (1991). Evolution of oviposition behaviour and host
preference in the lepidoptera. Annual Review of Entomolgy 36: 65-89.
Torres A.M., T. Mau-Lastovicka and R. Rezaaiyan, (1987). Total phenolics and
high-performance liquid chromatography of phenolic acids of avocado. Journal of
Agricultural Food Chemistry 35: 921-925.
157
Torto B., A. Hassanalli, K.N. Saxena and S. Nokoe, (1991). Feeding response of
Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) larvae to sorghum plant
phenolics and their analogs. Journal of Chemical Ecology 17: 67-68.
van den Berg J. and A.A. Ebenebe, (2001). Integrated management of stemborers in
Lesotho. Insect science and its Application 21: 389-394.
Vander Meer R.K., C.S. Lofgren and J.A. Seawright, (1995). Specificity of the red
imported fire ant (Hymenoptera: Formicidae) phagostimulant response to
carbohydrates. Florida Entomologist 78: 144-154.
Van Loon J.J.A., (1990). Chemoreception of phenolic acids and flavenoids in larvae
of two species of Pieris. Journal of Comparative Physiology A 166: 889-899.
Van Loon J.J.A., (1996). Chemosensory basis of oviposition and feeding behaviour
in herbivorous insects. Entomologia Experimentalis et Applicata 80: 7-13.
van Rensburg J.B.J., M.C. Walters and J.H. Giliomee, (1989). Selective oviposition
by the maize stalk borer, Busseola fusca (Fuller). Journal of the Entomological
Society of Southern Africa 52: 105–108.
Via, S. (1991). Specialised host plant performance of pea aphid clones is not altered
by experience. Ecology 72: 1420-1427.
158
Yazawa M., (1997). Characteristics of sucrose formation and the influence of UV
radiation on the feeding by the silkworm in leaves of the mulberry. National
Institute of Sericultural and Entomological Science 18: 1-77.
Yu S., (1989). Beta-glucosidase in four phytophagous Lepidoptera. Insect
Biochemistry 19:103.
Zibaee A. A.R. Bandani, M. Kafil, and S. Ramzi, (2008). Characterisation of α-
amylase in the midgut and the salivary glands of rice striped stem borer, Chilo
suppressalis Walker ( Lepidoptera: pyralidae). Journal of Asia-Pacific Entomology
11 201-205.
Zibaee A. A.R. Bandani, and S. Ramzi, (2009).Enzymatic properties of α- and β-
glucosidases extracted from midgut and salivary glands of rice striped stemborer,
Chilo suppressalis Walker ( Lepidoptera: Pyralidae).C. R. Biologies 332: 633-641
159
7. 0: APPENDICES
A: Representative HPLC Chromagram obtained following injection of 2 µL
solution of 10mg/ml dried Zea mays methanol extracts reconstituted in water
into a supelcosil LC-NH2 column in Shimdzu class-VP autosampler machine.
160
B: Representative HPLC Chromagram obtained following injection of 2 µL solution
of 50mg/ml dried Sorghum arundinaceum methanol extracts reconstituted in water
into a supelcosil LC-NH2 column in Shimdzu class-VP autosampler machine.
161
C: Representative HPLC Chromagram obtained following injection of 2 µL solution
of 50mg/ml dried Setaria megaphylla methanol extracts reconstituted in water into a
supelcosil LC-NH2 column in Shimdzu class-VP autosampler machine.
162
D: Representative HPLC Chromagram obtained following injection of 2 µL solution
of 50mg/ml dried Panicum desteum methanol extracts reconstituted in water into a
supelcosil LC-NH2 column in Shimdzu class-VP autosampler machine.
163
E: Representative HPLC Chromagram obtained following injection of 2 µL solution
of 50mg/ml dried Panicum maximum methanol extracts reconstituted in water into a
supelcosil LC-NH2 column in Shimdzu class-VP autosampler machine.
164
F: Representative HPLC Chromagram obtained following injection of 2 µL solution
of 50mg/ml dried Arundo donax methanol extracts reconstituted in water into a
supelcosil LC-NH2 column in Shimdzu class-VP autosampler machine.
165
G: Representative HPLC Chromagram obtained following injection of 2 µL solution
of 50mg/ml dried Peniseteum purpureum methanol extracts reconstituted in water
into a supelcosil LC-NH2 column in Shimdzu class-VP autosampler machine.